Phosphodiesterase inhibitor treatment

ABSTRACT

Methods and compositions are disclosed for the treatment of diseases or conditions produced by or associated with low cyclic nucleotide levels. The compositions comprise phosphodiesterase inhibitors and are formulated for intranasal and pulmonary administration.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.13/421,277, filed Mar. 15, 2012, which is a division of U.S. applicationSer. No. 12/508,530, filed Jul. 23, 2009, which claims the benefit ofU.S. Provisional Application No. 61/083,147, filed Jul. 23, 2008, eachof which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Phosphodiesterases (PDE) are a diverse family of enzymes that hydrolysecyclic nucleotides resulting in the modulation of intracellular levelsof the second messangers cAMP and cGMP, and hence, cell function.Numerous diseases and conditions result from low levels of cyclicnucleotides. The use of PDE inhibitors to raise cellular levels ofcyclic nucleotides offers the ability to prevent, treat, or amelioratediseases, conditions or their symptoms, however, systemic administrationmay not achieve therapeutically effective concentrations due tounacceptable side effects or to inability to obtain therapeutic levelsin clinically responsive tissues. There is a need for suitablecompositions and methods of delivery to achieve medically relevantconcentrations of PDE inhibitors without unacceptable side effects. Thepresent invention addresses these unmet needs.

SUMMARY OF INVENTION

In one aspect of the invention, a method is provided for treatinganosmia, hyposmia, ageusia, or hypogeusia comprising administering byintranasal administration to a patient, an effective amount of aphosphodiesterase (PDE) inhibitor that treats the patient's anosmia,hyposmia, ageusia, or hypogeusia. In one embodiment, the PDE inhibitoris a non-selective PDE inhibitor, PDE-1 selective inhibitor, PDE-2selective inhibitor, PDE-3 selective inhibitor, PDE-4 selectiveinhibitor, or a PDE-5 selective inhibitor. In another embodiment, thenon-selective PDE inhibitor is a methylxanthine derivative. In a furtherembodiment the methylxanthine derivative is caffeine, theophylline, IBMX(3-isobutyl-1-methylxanthine) aminophylline, doxophylline,cipamphylline, neuphylline, pentoxiphylline, or diprophylline. In aparticular embodiment, the methylxanthine derivative is theophylline orpentoxiphylline. In one embodiment the PDE-3 inhibitor is cilostazol.

In some embodiments, the effective amount of the PDE inhibitor is lessthan 2 mg. In other embodiments, the effective amount is less than 1 mg,500 μg, 250μ, or 100 μg. In one embodiment, the effective amount is 40μg.

In some embodiments, the PDE inhibitor is formulated as a liquid, whilein other embodiments it is formulated as a dry powder. In someembodiments, the PDE inhibitor is administered as a liquid, gel,ointment, cream, spray, aerosol, or dry powder. In other embodiments,the PDE inhibitor is administered at least once, twice, or thrice daily.

In some embodiments, successful treatment of anosmia or hyposmiaincreases a patient's taste or smell acuity by at least 5%. In otherembodiments, taste or smell acuity is measured objectively, while inother embodiments, acuity is measured subjectively. In some embodiments,the increase in taste or smell acuity is accompanied by an increase innasal mucus or saliva cAMP or cGMP levels. In further embodiments, theincrease in salivary or nasal mucus cAMP or cGMP level is at least 10%over the untreated level.

In one aspect of the invention, a method is provided for treatinganosmia or hyposmia comprising administering to a patient in need, aneffective amount of a PDE inhibitor, wherein the blood concentration ofthe PDE inhibitor does not exceed 1 mg/dl.

In another aspect of the invention, a method is provided for increasingnasal mucus or saliva cyclic adenosine 3′, 5′-monophosphate (cAMP) orcyclic guanosine 3′, 5′-monophosphate (cGMP) levels comprisingadministering by intranasal administration to a patient in need, aneffective amount of a phosphodiesterase (PDE) inhibitor, whereby thecAMP or cGMP levels are increased at least 10% over the untreated level.

In one aspect, a method is provided for increasing taste or smell acuitycomprising administering to a patient in need, an effective amount of aPDE inhibitor, wherein the PDE inhibitor is administered by intranasaladministration and wherein taste or smell acuity is increased. In someembodiments, taste or smell acuity is increased at least 5% or 10% overpre-treatment levels. In some embodiments, taste or smell acuity ismeasured objectively, while in other embodiments, it is subjectively. Infurther embodiments, the increase in taste or smell acuity isaccompanied by an increase in nasal mucus or saliva cAMP or cGMP levels.In some embodiments, nasal mucus or saliva cAMP or cGMP levels increaseat least 10% compared to the untreated state.

In another aspect, a method is provided for compensating for apathologic rate of cAMP or cGMP metabolism comprising administering to apatient in need, an effective amount of a PDE inhibitor, wherein the PDEinhibitor is administered by intranasal administration and wherein cAMPor cGMP metabolism is decreased. In some embodiments, the bloodconcentration of the PDE inhibitor does not exceed 1 mg/dl.

In one aspect of the invention, a method is provided for screeningpatients for suitability for PDE inhibitor therapy for anosmia,hyposmia, ageusia or hypogeusia by administering to a patient achallenge dose of a PDE inhibitor; determining the nasal mucus orsalivary level of cGMP; and comparing the patient's cGMP level to athreshold value, wherein patients who have a cGMP level equal to orgreater than the threshold value are candidates for PDE inhibitortherapy to treat anosmia, hyposmia, ageusia or hypogeusia.

In another aspect of the invention, a pharmaceutical composition forintranasal administration is provided comprising a phosphodiesterase(PDE) inhibitor, wherein the composition comprises less than 1 mg of thePDE inhibitor and a pharmaceutically acceptable carrier. In someembodiments, the PDE inhibitor is selected from the group consisting ofnon-selective PDE inhibitors, PDE-1 selective inhibitors, PDE-2selective inhibitors, PDE-3 selective inhibitors, PDE-4 selectiveinhibitors, and PDE-5 selective inhibitors. In some embodiments, the PDEinhibitor is a non-selective PDE inhibitor, while in other embodiments,the non-selective PDE inhibitor is a methylxanthine derivative. Infurther embodiments, the methylxanthine derivative is caffeine,theophylline, IBMX (3-isobutyl-1-methylxanthine) aminophylline,doxophylline, cipamphylline, neuphylline, pentoxiphylline, ordiprophylline. In one embodiment, the methylxanthine derivative istheophylline or pentoxiphylline. In a further embodiment, the PDEinhibitor is a PDE-3 selective inhibitor. In a still further embodiment,the PDE-3 selective inhibitor is cilostazol.

In some embodiments the composition is a dry powder. In otherembodiments, the dry powder composition further comprises an excipient.In further embodiments, the composition is a liquid. In someembodiments, the liquid composition further comprises an excipient. Infurther embodiments, the excipient is a preservative.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates the structure of the patient flow of the clinicaltrial showing the number and percentage of patients that returned ontheophylline treatment. Three hundred twelve patients began the study on200 mg. If improved <5% patient number indicates progression to nextstep up in dose (400 mg, 600 mg, 800 mg). Left sided numbers (lines)indicate numbers of distant patients who returned for first time (seetext). Right sided numbers (lines) indicate patient numbers who improved<5% and returned on a higher dose. Difference between right sidednumbers and patient numbers who improved <5% (in right boxes) indicatenumber of patient drop outs at each dose. If improved ≥5% patient datanot included in further step-up doses.

FIG. 2A-B is a comparison of DT and RT values for pyridine (PYRD),nitrobenzene (NO2B), thiophene (THIO) and amyl acetate (AA) in 312patients before treatment and in all patients in each group aftertreatment with oral theophylline at 200 mg, 400 mg, 600 mg and 800 mg(see Tables II, IV-VII).

FIG. 3A-B is a comparison of ME and H values for pyridine (PYRD),nitrobenzene (NO2B), thiophene (THIO) and amyl acetate (AA) in 312patients before treatment and in all patients in each group aftertreatment with oral theophylline at 200 mg, 400 mg, 600 mg and 800 mg(see Tables II, IV-VII).

DETAILED DESCRIPTION OF THE INVENTION

Numerous PDEs are known with notable PDEs, their inhibitors and uses forthese inhibitors listed below.

Theophylline and papaverine are representative members of non-specificPDE inhibitors that are prescribed orally to treat asthma and chronicobstructive pulmonary disease (COPD) through the relaxation of smoothmuscle in the airways. Theophylline has anti-inflammatory effects on theairways that is useful to combat the abnormal inflammation seen inasthmatics. Most importantly, this anti-inflammatory effect is found atlevels in the blood well below that which causes the common side effectsseen in most people. Patients with emphysema and chronic bronchitis canalso be helped with theophylline when their symptoms are partiallyrelated to reversible airway narrowing.

Theophylline is a methylxanthine derivative; other non-selectivephosphodiesterase inhibitors in this class include caffeine, IBMX(3-isobutyl-1-methylxanthine, aminophylline, doxophylline,cipamphylline, theobromine, pentoxifylline (oxpentifylline) anddiprophylline.

PDE1 selective inhibitors formerly known as calcium- andcalmodulin-dependent phosphodiesterases includeeburnamenine-14-carboxylic acid ethyl ester (vinpocetine), used toinduce vasorelaxtion on cerebral smooth muscle tissue.

PDE2 decreases aldosterone secretion and is suggested to play animportant role in the regulation of elevated intracellularconcentrations of cAMP and cGMP in platelets. Several regions of thebrain express PDE2 and rat experiments indicate that inhibition of PDE2enhances memory. PDE2 may play a role in regulation of fluid and cellextravasation during inflammatory conditions as PDE2 is localized tomicrovessels, especially venous capillary and endothelial cells, butapparently not to larger vessels. PDE2 may also be a goodpharmacological target for pathological states such as sepsis or in morelocalized inflammatory responses such as thrombin-induced edemaformation in the lung. PDE-2 selective inhibitors include EHNA(erythro-9-(2-hydroxy-3-nonyl) adenine),9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one (PDP),and BAY 60-7750.

The PDE3 family hydrolyzes cAMP and cGMP, but in a manner suggestingthat in vivo, the hydrolysis of cAMP is inhibited by cGMP. They also aredistinguished by their ability to be activated by severalphosphorylation pathways including the PKA and PI3K/PKB pathways. PDE3Ais relatively highly expressed in platelets, as well as in cardiacmyocytes and oocytes. PDE3B is a major PDE in adipose tissue, liver, andpancreas, as well as in several cardiovascular tissues. Both PDE3A andPDE3B are highly expressed in vascular smooth muscle cells and arelikely to modulate contraction.

PDE3 inhibitors mimic sympathetic stimulation to increase cardiacinotropy, chronotropy and dromotropy. PDE3 inhibitors also antagonizeplatelet aggregation, increase myocardial contractility, and enhancevascular and airway smooth muscle relaxation. PDE3A is a regulator ofthis process and PDE3 inhibitors effectively prevent aggregation. Infact one drug, cilastazol (Pletal), is approved for treatment ofintermittent claudication. Its mechanism of action is thought to involveinhibition of platelet aggregation along with inhibition of smoothmuscle proliferation and vasodilation. PDE3-selective inhibitors includeenoximone, milrinone (Primacor), amrinone, cilostamide, cilostazol(Pletal) and trequinsin.

PDE4 inhibitors can effectively suppress release of inflammatorymediators e.g., cytokines, inhibit the production of reactive oxygenspecies and immune cell infiltration. PDE4-selective inhibitors includemesembrine, rolipram, Ibudilast, a neuroprotective and bronchodilatordrug used mainly in the treatment of asthma and stroke, and roflumilast(Daxas) and cilomilast (Airflo), currently in phase III clinical trialsfor treatment of chronic obstructive pulmonary disease. Otherinflammatory diseases for which PDE4 inhibitors are currently beingdeveloped include asthma, arthritis, and psoriasis.

PDE5 is a regulator of vascular smooth muscle contraction best known asthe molecular target for several well-advertised drugs used to treaterectile dysfunction and pulmonary hypertension. In the lung, inhibitionof PDE5 opposes smooth muscle vasoconstriction, and PDE5 inhibitors arein clinical trials for treatment of pulmonary hypertension.

PDE5-selective inhibitors include Sildenafil, tadalafil, vardenafil,udenafil and avanafil.

PDE inhibitors inhibit cellular apoptosis by inhibiting TNF alpha, TRAILand their metabolites. PDE inhibitors activate the production andsecretion of nitric oxide in all tissues thereby inducing vasorelaxationor vasodilation of all blood vessels including those of the peripheralblood vessels (inhibiting intermittent claudication), the distalextremities and in the penile region contributing to penile erection.

PDE inhibitors useful in the present invention include, for example,filaminast, piclamilast, rolipram, Org 20241, MCI-154, roflumilast,toborinone, posicar, lixazinone, zaprinast, sildenafil,pyrazolopyrimidinones (such as those disclosed in WO 98/49166),motapizone, pimobendan, zardaverine, siguazodan, CI-930, EMD 53998,imazodan, saterinone, loprinone hydrochloride, 3-pyridinecarbonitrilederivatives, denbufyllene, albifylline, torbafylline, doxofylline,theophylline, pentoxofylline, nanterinone, cilostazol, cilostamide, MS857, piroximone, milrinone, aminone, tolafentrine, dipyridamole,papaverine, E4021, thienopyrimidine derivatives (such as those disclosedin WO 98/17668), triflusal, ICOS-351,tetrahydropiperazino[1,2-b]beta-carboline-1,4-dione derivatives (such asthose disclosed in U.S. Pat. No. 5,859,006, WO 97/03985 and WO97/03675), carboline derivatives, (such as those disclosed in WO97/43287), 2-pyrazolin-5-one derivatives (such as those disclosed inU.S. Pat. No. 5,869,516), fused pyridazine derivatives (such as thosedisclosed in U.S. Pat. No. 5,849,741), quinazoline derivatives (such asthose disclosed in U.S. Pat. No. 5,614,627), anthranilic acidderivatives (such as those disclosed in U.S. Pat. No. 5,714,993),imidazoquinazoline derivatives (such as those disclosed in WO 96/26940),and the like. Also included are those phosphodiesterase inhibitorsdisclosed in WO 99/21562 and WO 99/30697. The disclosures of each ofwhich are incorporated herein by reference in their entirety. In someembodiments, the intranasal composition does not comprise a PDE5selective inhibitor.

Sources of information for the above, and other phosphodiesteraseinhibitors include Goodman and Gilman, The Pharmacological Basis ofTherapeutics (9th Ed.), McGraw-Hill, Inc. (1995), The Physician's DeskReference (49th Ed.), Medical Economics (1995), Drug Facts andComparisons (1993 Ed), Facts and Comparisons (1993), and The Merck Index(12th Ed.), Merck & Co., Inc. (1996), the disclosures of each of whichare incorporated herein by reference in their entirety.

The following definitions are used throughout the specification.

“Phosphodiesterase inhibitor” or “PDE inhibitor” refers to any compoundthat inhibits a phosphodiesterase enzyme, isozyme or allozyme. The termrefers to selective or non-selective inhibitors of cyclic guanosine3′,5′-monophosphate phosphodiesterases (cGMP-PDE) and cyclic adenosine3′,5′-monophosphate phosphodiesterases (cAMP-PDE).

“Patient” refers to animals, preferably mammals, more preferably humans.

Other medicaments may be combined with or administered contemporaneouslywith at least one PDE inhibitors to complement and/or to enhance theprevention or treatment effect of a PDE inhibitor. These othermedicaments include vasoactive agents, anticholinergic agents,leukotriene receptor antagonists, thromboxane synthetase inhibitors,thromboxane A2 receptor antagonist, mediator release inhibitor,antihistamic agent, cytokine inhibitor, prostaglandins, forskolin,elastase inhibitor, steroid, expectorant, or antibacterial agent. Theother medicaments can be administered simultaneously with, subsequentlyto, or prior to administration of the PDE inhibitors.

In one embodiment, the patient is administered a therapeuticallyeffective amount of a PDE inhibitor and a vasoactive agent. A vasoactiveagent is any therapeutic agent capable of relaxing vascular smoothmuscle. Suitable vasoactive agents include, but are not limited to,potassium channel activators (such as, for example, nicorandil,pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam and the like);calcium blockers (such as, for example, nifedipine, veraparmil,diltiazem, gallopamil, niludipine, nimodipins, nicardipine, and thelike); beta-blockers (such as, for example, butixamine,dichloroisoproterenol, propanolol, alprenolol, bunolol, nadolol,oxprenolol, perbutolol, pinodolol, sotalol, timolol, metoprolol,atenolol, acebutolol, bevantolol, pafenolol, tolamodol, and the like);long and short acting alpha-adrenergic receptor antagonist (such as, forexample, phenoxybenzamide, dibenamine, doxazosin, terazosin,phentolamine, tolazoline, prozosin, trimazosin, yohimbine, moxisylyteand the like adenosine, ergot alkaloids (such as, for example,ergotamine, ergotamine analogs, including, for example, acetergamine,brazergoline, bromerguride, cianergoline, delorgotrile, disulergine,ergonovine maleate, ergotamine tartrate, etisulergine, lergotrile,lysergide, mesulergine, metergoline, metergotamine, nicergoline,pergolide, propisergide, proterguride, terguride); vasoactive intestinalpeptides (such as, for example, peptide histidine isoleucine, peptidehistidine methionine, substance P, calcitonin gene-related peptide,neurokinin A, bradykinin, neurokinin B, and the like); dopamine agonists(such as, for example, apomorphine, bromocriptine, testosterone,cocaine, strychnine, and the like); opioid antagonists (such as, forexample, naltrexone, and the like); prostaglandins (such as, forexample, alprostadil, prostaglandin E2, prostaglandin F2, misoprostol,enprostil, arbaprostil, unoprostone, trimoprostil, carboprost,limaprost, gemeprost, lantanoprost, omoprostil, beraprost, sulpostrone,rioprostil, and the like); endothelin antagonists (such as, for example,bosentan, sulfonamide endothelin antagonists, BQ-123, SQ 28608, and thelike) and mixtures thereof.

In one aspect of the present invention, methods are provided to preventor treat diseases associated with or caused by the increased(pathological) metabolism of cyclic adenosine 3′,5′-monophosphate (cAMP)or cyclic guanosine 3′,5′-monophosphate (cGMP), including, for example,anosmia, hyposmia, ageusia, hypogeusia, hypertension, pulmonaryhypertension, congestive heart failure, renal failure, myocardialinfraction, stable, unstable and variant (Prinzmetal) angina,atherosclerosis, cardiac edema, renal insufficiency, nephrotic edema,hepatic edema, stroke, asthma, bronchitis, chronic obstructive pulmonarydisease (COPD), cystic fibrosis, dementia including Alzheimer's disease,immunodeficiency, premature labor, Parkinson's disease, multiplesclerosis, dysmenorrhoea, benign prostatic hyperplasis (BPH), bladderoutlet obstruction, incontinence, conditions of reduced blood vesselpatency, e.g., postpercutaneous transluminal coronary angioplasty(post-PTCA), peripheral vascular disease, allergic rhinitis, glaucoma,malignancies and diseases characterized by disorders of gut motility,e.g, irritable bowel syndrome (IBS), rheumatoid arthritis, systemiclupus erythematosus, psoriasis, and other autoimmune diseases,Huntington's chorea, and Amyotrophic lateral sclerosis (ALS), byadministering to a patient in need thereof a therapeutically effectiveamount of the compounds and/or compositions described herein.

In some embodiments, the intranasal, pulmonary or lingual administrationof a PDE inhibitor can increase cell, tissue or organ levels of cAMP orcGMP. In some embodiments, the increase in cAMP or cGMP levels is atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%,400%, 500% or 1000% over the untreated state. In other embodiments, cAMPor cGMP levels are increased to at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, or 500% of the levelsseen in controls, i.e., normal individuals. In some embodiments of theinvention, a method is provided for increasing nasal mucus or salivarycAMP or cGMP levels, wherein an effective amount of a PDE inhibitor isadministered intranasally to a patient resulting in an increase in thecAMP or cGMP levels of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 150%, 200%, 300%, 400%, 500%, or 1000% over the untreatedlevel. In other embodiments, nasal mucus or salivary cAMP or cGMP levelsare increased to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 125%, 150%, 200%, 300%, 400%, or 500% of the levels seen in normalindividuals.

In some embodiments, the administration of an effective amount of a PDEinhibitor by intranasal, lingual or pulmonary administration does notproduce a detectable blood level of the PDE inhibitor. In someembodiments, the administration of an effective amount of a PDEinhibitor by intranasal, lingual or pulmonary administration producesblood concentration of the PDE inhibitor that are less than 5 mg/dl, 2mg/dl, 1 mg/dl, 500 μg/dl, 250 μg/dl, 100 μg/dl, 50 μg/dl, 25 μg/dl, 10μg/dl, 5 μg/dl, or 1 μg/dl.

In some embodiments, intranasal or lingual administration of aneffective amount of a PDE inhibitor increases taste or smell acuity. Insome embodiments, the increase in taste or smell acuity is at least 5%,10%, 20%, 30%, 40%, 50%, 75%, or 100% compared to the untreated state.In other embodiments, taste or smell acuity is increased to at least 5%,10%, 20%, 30%, 40%, 50%, 75%, or 100% of the acuity of normalindividuals. In some embodiments, taste or smell acuity is measuredobjectively, while in other embodiments taste or smell acuity ismeasured subjectively.

When administered in vivo, the compounds and compositions of the presentinvention can be administered in combination with pharmaceuticallyacceptable carriers and in dosages described herein. The compounds andcompositions of the present invention can be formulated aspharmaceutically acceptable neutral (free base) or salt forms.Pharmaceutically acceptable salts include, for example, those formedwith free amino groups such as those derived from hydrochloric,hydrobromic, hydroiodide, phosphoric, sulfuric, acetic, citric, benzoic,fumaric, glutamic, lactic, malic, maleic, succinic, tartaric,p-toluenesulfonic, methanesulfonic acids, gluconic acid, and the like,and those formed with free carboxyl groups, such as those derived fromsodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

“Therapeutically effective amount” refers to the amount of a PDEinhibitor with or without additional agents that is effective to achieveits intended purpose. While individual patient needs may vary,determination of optimal ranges for effective amounts of each of thecompounds and compositions is within the skill of an ordinarypractitioner of the art. Generally, the dosage required to provide aneffective amount of the composition, and which can be adjusted by one ofordinary skill in the art, will vary, depending on the age, health,physical condition, sex, weight, extent of the dysfunction of therecipient, frequency of treatment and the nature and scope of thedysfunction.

The amount of a given PDE inhibitor which will be effective in theprevention or treatment of a particular dysfunction or condition willdepend on the nature of the dysfunction or condition, and can bedetermined by standard clinical techniques, including reference toGoodman and Gilman, supra; The Physician's Desk Reference, supra;Medical Economics Company, Inc., Oradell, N.J., 1995; and Drug Facts andComparisons, Inc., St. Louis, Mo., 1993. The precise dose to be used inthe formulation will also depend on the route of administration, and theseriousness of the dysfunction or disorder, and should be decided by thephysician and the patient's circumstances.

The nasal and/or pulmonary administered PDE inhibitors can be used atdose ranges and over a course of dose regimen that are the same orsubstantially equivalent to those used for oral administration. Thenasal and/or pulmonary administered PDE inhibitors can also be used inlower doses and in less extensive regimens of treatment. The amount ofactive ingredient that can be combined with one or more carrier materialto produce a single dosage form will vary depending upon the hosttreated and the particular mode of administration.

Representative daily intranasal, lingual or pulmonary dosages arebetween about 1.0 μg and 2000 mg per day, between about 1.0 μg and 500.0mg per day, between about 10 μg and 100.0 mg per day, between about 10μg and about 10 mg per day, between about 10 μg and 1.0 mg per day,between about 10 μg and 500 μg per day or between about 1 μg and 50 μgper day of the active ingredient comprising a preferred compound. Theseranges of dosage amounts represent total dosage amounts of the activeingredient per day for a given patient. In some embodiments, the dailyadministered dose is less than 2000 mg per day, 1000 mg per day, 500 mgper day, 100 mg per day, 10 mg per day, 1.0 mg per day, 500 μg per day,300 μg per day, 200 μg per day, 100 μs per day or 50 μg per day. Inother embodiments, the daily administered dose is at least 2000 mg perday, 1000 mg per day, 500 mg per day, 100 mg per day, 10 mg per day, 1.0mg per day, 500 μg per day, 300 μg per day, 200 μg per day, 100 μs perday or 50 μg per day. In some embodiments, on a per kilo basis, suitabledosage levels of the compounds will be between about 0.001 μg/kg andabout 10.0 mg/kg of body weight per day, between about 0.5 μg/kg andabout 0.5 mg/kg of body weight per day, between about 1.0 μg/kg andabout 100 μg/kg of body weight per day, and between about 2.0 μg/kg andabout 50 μg/kg of body weight per day of the active ingredient. In otherembodiments, the suitable dosage level on a per kilo basis is less than10.0 mg/kg of body weight per day, 1 mg/kg of body weight per day, 500μg/kg of body weight per day, 100 μg/kg of body weight per day, 10 μg/kgof body weight per day of the active ingredient, or 1.0 μg/kg of bodyweight per day of active ingredient. In further embodiments, thesuitable dosage level on a per kilo basis is at least 10.0 mg/kg of bodyweight per day, 1 mg/kg of body weight per day, 500 μg/kg of body weightper day, 100 μg/kg of body weight per day, 10 μg/kg of body weight perday of the active ingredient, or 1.0 μg/kg of body weight per day ofactive ingredient.

In some embodiments, the individual or single intranasal, lingual and/orpulmonary dose of the PDE inhibitors is less than 10 mg, less than 2 mg,less than 1 mg, less than 500 less than 200 μg, less than 100 μg, orless than 50 μg per dosage unit or application. In other embodiments,the individual or single intranasal, lingual and/or pulmonary dose ofthe PDE inhibitors is at least 10 mg, 1 mg, 500 μg, 200 μg, 100 μg, 50μg per dosage unit or application. In further embodiments, theindividual or single intranasal, lingual and/or pulmonary dose of thePDE inhibitors ranges from 1 μg to 10 mg, 10μ to 1 mg, 10 μg to 500 μg,10 μg to 250 μg, 10 μg to 200 μg, 10 μg to 100 μg, 10 μg to 50 μg, 25 μgto 100 μg, 25 μg to 250 μg, 50 μg to 500 or 100 μg to 1.0 mg

The number of times per day that a dose is administered will depend uponsuch pharmacological and pharmacokinetic factors as the half-life of theactive ingredient, which reflects its rate of catabolism and clearance,as well as the minimal and optimal blood plasma or other body fluidlevels of said active ingredient attained in the patient which arerequired for therapeutic efficacy. Typically, the PDE inhibitors aregiven once, twice, trice, or four times daily. PDE inhibitors may alsobe administered on a less frequent basis, such as every other day, everythree, four, five, six or seven days.

Other factors may also be considered in deciding upon the number ofdoses per day and the amount of active ingredient per dose to beadministered. Not the least important of such other factors is theindividual response of the patient being treated. Thus, for example,where the active ingredient is used to treat or prevent asthma, and isadministered loco-regionally via aerosol inhalation into the lungs, fromone to four doses consisting of actuations of a dispensing device, i.e.,“puffs” of an inhaler, may be administered. each day, with each dosecontaining from about 10.0 to about 10.0 mg of active ingredient.

Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems and are in the same ranges orless than as described for the commercially available compounds in thePhysician's Desk Reference, supra.

Aerosols

By “aerosol” is meant any composition of a PDE inhibitor administered asan aerosolized formulation, including for example an inhalation spray,inhalation solution, inhalation suspension, a nebulized solution, ornasal spray. Aerosolized formulations can deliver high concentrations ofa PDE inhibitor directly to the airways with low systemic absorption.Solutions for aerosolization typically contain at least onetherapeutically active PDE inhibitor dissolved or suspended in anaqueous solution that may further include one or more excipients (e.g.,preservatives, viscosity modifiers, emulsifiers, or buffering agents).The solution acts as a carrier for the PDE inhibitor. In someembodiments, the preservative is methylparaben or propylparaben. Theseformulations are intended for delivery to the respiratory airways byinspiration.

A major limitation of pulmonary delivery is the difficulty of reachingthe deep lung. To achieve high concentrations of a PDE inhibitorsolution in both the upper and lower respiratory airways, the PDEinhibitor solution is preferably nebulized in jet nebulizers, aultrasonic nebulizer, or an electronic nebulizer particularly thosemodified with the addition of one-way flow valves, such as for example,the Pan LC Plus™ nebulizer, commercially available from Pari RespiratoryEquipment, Inc., Richmond, Va., which delivers up to 20% more drug thanother unmodified nebulizers.

The pH of the formulation is also important for aerosol delivery. Whenthe aerosol is acidic or basic, it can cause bronchospasm and cough. Thesafe range of pH is relative and depends on a patient's tolerance. Somepatients tolerate a mildly acidic aerosol, which in others will causebronchospasm. Typically, an aerosol solution having a pH less than 4.5induces bronchospasm. An aerosol solution having pH between 4.5 and 5.5will occasionally cause this problem. The aerosol solution having a pHbetween 5.5 and 7.0 is usually considered safe. Any aerosol having pHgreater than 7.0 is to be avoided as the body tissues are unable tobuffer alkaline aerosols and result in irritation and bronchospasm.Therefore, the pH of the formulation is preferably maintained between4.5 and 7.0, more preferably between 5.0 and 7.0 and most preferablybetween 5.5 and 6.5 to permit generation of a PDE inhibitor aerosol thatis well tolerated by patients without any secondary undesirable sideeffects such as bronchospasm and cough. The osmolarity of theformulation can also be adjusted to osmolarities of about 250 to 350mosm/L, according to the patient's tolerance.

Drops and Gels

In some embodiments, the PDE inhibitor is directly applied to the nasalor lingual epithelium as a liquid, cream, lotion, ointment or gel. Thesefluids or semifluids contain at least one therapeutically active PDEinhibitor and may further include at least one excipient (e.g.,preservatives, viscosity modifiers, emulsifiers, or buffering agents)that are formulated for administration as nose drops, or applied with anapplicator to the inside of the nasal passages. In some embodiments, thepreservative is methylparaben or propylparaben. The pH of theformulation is preferably maintained between 4.5 and 7.0, morepreferably between 5.0 and 7.0 and most preferably between 5.5 and 6.5.The osmolarity of the formulation can also be adjusted to osmolaritiesof about 250 to 350 mosm/L.

Dry Powder Formulation

As an alternative therapy to aerosol, liquid or gel delivery, the PDEinhibitor may be administered in a dry powder formulation forefficacious delivery into the nasal cavity and/or endobronchial space.Dry powder formulation is convenient because it does not require furtherhandling by a physician, pharmacist or patient such as diluting orreconstituting the agent as is often required with nebulizers.Furthermore, dry powder delivery devices are sufficiently small andfully portable. Dry powder formulations may also be applied directly onthe lingual epithelium.

For dry powder formulations, a PDE inhibitor and/or carrier is processedto median diameter ranging from 0.001-250 μm typically by media milling,jet milling, spray drying, super-critical fluid energy, or particleprecipitation techniques. Particles of a desired size ranges can also beobtained through the use of sieves. Frequently, milled particles arepassed through one or more sieves to isolate a desired size range. Insome embodiments intended for pulmonary administration, the PDEinhibitor and/or carrier has a median diameter ranging from 0.01-25 μm,0.1-10 μm, 1-10 μm, 1-5 μm, or 2-5 μm. In further embodiments intendedfor pulmonary administration, the PDE inhibitor and/or carrier has amedian diameter ranging less than 20 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm,or 1 μm. In some embodiments intended for nasal administration, the PDEinhibitor and/or carrier has a median diameter ranging from 1-250 μm,5-200 μm, 10-150 μm, 10-100 μm, 10-50 μm, 15-100 μm, 15-50 μm, or 20-60μm. In further embodiments intended for nasal administration, the PDEinhibitor and/or carrier has a median diameter of less than 250 μm, 200μm, 150 μm, 100 μm, 75 μm, 60 μm, 50 μm, 40 μm or 30 μm. In otherembodiments intended for nasal administration, the PDE inhibitor and/orcarrier has a median diameter of at least 20 μm, 30 μm, 40 μm, 50 μm, 60μm, 75 μm, 100 μm, 150 μm or 200 μm.

In some embodiments, a pharmaceutically acceptable carrier for thepresent compositions and formulations include but are not limited toamino acids, peptides, proteins, non-biological polymers, biologicalpolymers, simple sugars, carbohydrates, gums, inorganic salts and metalcompounds which may be present singularly or in combination. In someembodiments, the pharmaceutically acceptable carrier comprises native,derivatized, modified forms, or combinations thereof.

In some embodiments, useful proteins include, but are not limited to,gelatin or albumin. In some embodiments, useful sugars that can serve aspharmaceutically acceptable carriers include, but are not limited tofructose, galactose, glucose, lactitol, lactose, maltitol, maltose,mannitol, melezitose, myoinositol, palatinite, raffinose, stachyose,sucrose, trehalose, xylitol, hydrates thereof, and combinations ofthereof.

In some embodiments, useful carbohydrates that can serve aspharmaceutically acceptable carriers include, but are not limited tostarches such as corn starch, potato starch, amylose, amylopectin,pectin, hydroxypropyl starch, carboxymethyl starch, and cross-linkedstarch. In other embodiments, useful carbohydrates that can serve aspharmaceutically acceptable carriers include, but are not limited tocellulose, crystalline cellulose, microcrystalline cellulose,α-cellulose, methylcellulose, hydroxypropyl cellulose, carboxymethylcellulose, ethyl cellulose, hydroxypropyl methyl cellulose, andcellulose acetate.

In some embodiments, the composition or formulation includes anexcipient. Useful excipients include, but are not limited to,fluidizers, lubricants, adhesion agents, surfactants, acidifying agents,alkalizing agents, agents to adjust pH, antimicrobial preservatives,antioxidants, anti-static agents, buffering agents, chelating agents,humectants, gel-forming agents, or wetting agents. Excipients alsoinclude coloring agents, coating agents, sweetening, flavoring andperfuming and other masking agents. The compositions and formulations ofthis invention may include a therapeutic agent with an individualexcipient or with multiple excipients in any suitable combination, withor without a carrier.

The dry powder formulations of the present invention may be useddirectly in metered dose or dry powder inhalers. With dry powderinhalers, the inspiratory flow of the patient accelerates the powder outof the device and into the nasal and/or oral cavity. Alternatively, drypowder inhalers may employ an air source, a gas source, orelectrostatics, to deliver the therapeutic agent. The dry powderformulations are temperature stable and have a physiologicallyacceptable pH of 4.0-7.5, preferably 6.5 to 7.0.

Kits/Articles of Manufacture

For use of the therapeutic compositions described herein, kits andarticles of manufacture are also described. In some embodiments, suchkits include a carrier, package, or container that is compartmentalizedto receive one or more blister packs, bottles, tubes, capsules, and thelike. In certain embodiments, the pharmaceutical compositions arepresented in a pack or dispenser device which contains one or more unitdosage forms containing a compound provided herein. In otherembodiments, the pack contains metal or plastic foil, such as a blisterpack. In some embodiments, the pack contains capsules, vials, or tubes.In other embodiments, the pack or dispenser device is accompanied byinstructions for administration. In some embodiments, the dispenser isdisposable or single use, while in other embodiments, the dispenser isreusable. In certain embodiments, the pharmaceutical formulations arepreloaded into the device.

In still other embodiments, the pack or dispenser also accompanied witha notice as required by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals. This notice states thatthe drug is approved by the agency for human or veterinaryadministration. Such notice, for example, is the labeling approved bythe U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

The articles of manufacture provided herein may also contain anadministration or dispensing device. Examples of administration devicesinclude pulmonary inhalers and intranasal applicators. Pumps may beprovided with the inhalers and intranasal devices, or the pumps may bebuilt into the devices. Alternatively, a propellant may be included withor it may be stored within the devices.

Such kits optionally comprise an identifying description or label forthe containers. In further embodiments, the label is on a container withletters, numbers or other characters forming the label and attached,molded or etched into the container itself; a label is associated with acontainer when it is present within a receptacle or carrier that alsoholds the container, e.g., as a package insert. In some embodiments, alabel is used to indicate that the contents are to be used for aspecific therapeutic application. In yet other embodiments, the labelalso indicates directions for use of the contents, such as in themethods described herein. In some embodiments, a set of instructions mayalso be included, generally in the form of a package insert. Theinformational material may contain instructions on how to dispense thepharmaceutical composition, including description of the type ofpatients who may be treated, the schedule (e.g., dose and frequency),and the like.

The invention also relates to a set (kit) consisting of separate packsof kits that are frequently assembled for shipping or for patientconvenience, such as a weekly, biweekly or monthly supply of amedicament.

EXPERIMENTAL SECTION Example 1

Patients with smell loss (hyposmia) reflect a clinically diverse groupof patients (1-10). Whereas there is common agreement that many patientsexhibit this clinical problem, there is no agreement with respect totheir treatment. Indeed, most groups who evaluate these patientsconsider that there are few, if any, medically relevant treatments forthem.

In an attempt to elucidate the biochemical pathology of hyposmia, totalprotein fractionation was performed on saliva (14, 15) and nasal mucus(16), since these fluids bathe both taste buds and olfactory epithelialtissues, respectively, and contain substances which are critical tomaintain these sense organs (14-16). It was discovered that somepatients with smell loss had diminished salivary (17) and nasal mucus(18) levels of the saliva and nasal mucus protein carbonic anhydrase(CA) VI, a putative stem cell growth factor; treatment of these patientswith exogenous zinc increases both salivary and nasal mucus CA VI (19)leading to an increase in smell acuity (19). These CAVI deficientpatients, however, represent only a fraction of the total patient group(1, 2).

Further investigation revealed many of the non-CAVI deficient patientsexhibited lower than normal levels of both cAMP and cGMP in their saliva(31) and nasal mucus (32). When the cAMP and cGMP levels in the salivaand in the nasal mucus of these patients was compared against theseverity of their smell loss, it was noted that as smell loss severityincreased (worsened) levels of these cyclic nucleotides in saliva (34)and nasal mucus (35) decreased. Since these cyclic nucleotides act asgrowth factors for several neural tissues (36-38) including olfactorytissues (39-45), it was reasoned that lower than normal levels of cyclicnucleotides may play a role in generation of their hyposmia. To testthis hypothesis, patients were treated with the PDE inhibitortheophylline in an effort to increase saliva and nasal mucus levels ofthese cyclic nucleotides. It was found that hyposmia was corrected inmany of them as demonstrated by improvement of psychophysicalmeasurements of hyposmia, (1, 2, 49), by increased brain activation toseveral olfactory stimuli through measurements of functional magneticresonance imaging (fMRI) (48) and associated with changes in serumtheophylline (44).

In order to confirm these initial studies, theophylline was given to 312patients in a fixed design, controlled, open trial over a period ofseven years. These patients exhibited salivary (32) and nasal mucus (33)levels of cAMP and cGMP below the normal mean. Studies were approved byan Institutional Review Board and all patients gave informed consent.

The patients ranged in age from 18 to 86 y (55±1 y, mean±SEM) andconsisted of 178 women, aged 18-85 y (55±2 y) and 134 men, aged 23-86 y(54±3 y). Patients reported a history of smell loss extending from 2months to 40 y (6.5+1.0 y). Etiology of smell loss varied; the majorcauses of hyposmia were post influenza-like hyposmia [97 patients, 31.1%of the total (50)] and allergic rhinitis [97 patients, 31.1% of thetotal (51)] followed by head injury [42 patients, 13.5% (52)] andseveral other causes, as previously described [76 patients, 24.4% (1,2)]. Levels of CA VI in their saliva and nasal mucus were within normallevels.

Patients initially reported their sensory dysfunction as either loss oftaste (i.e., flavor) and/or smell function. This subjective response wasdocumented by objective psychophysical measurements of olfactoryfunction administered to each patient by use of a forced-choice,three-stimuli, stepwise-staircase technique in a fixed, controlleddesign (1, 53). Efficacy of this technique and results of testing werepreviously documented in a double-blind clinical trial (53). Four odorswere used; they were pyridine (dead-fish odor), nitrobenzene(bitter-almond odor), thiophene (petroleum-like odor) and amyl acetate(banana-oil odor). Detection thresholds (DT), recognition thresholds(RT) and magnitude estimation (ME) values for each odor were determinedas previously described (1, 53). Thresholds were converted into bottleunits (BU) as previously described (53) and results reported as M±SEM ofcorrect responses for each odor in each treatment group; ME was reportedin % and results calculated to obtain M±SEM for each treatment group forall correct responses using data for the four highest odorconcentrations presented (from 10^(−2M)—an absolute odor concentration).

In addition, each patient graded the hedonic (H) value of each odorpresented for these same odor concentrations (from 10^(−2M)—an absoluteodor concentration using a −100-0-+100 scale). If they considered thepresented odor pleasant (“they wished to smell the odor again”) theygraded the odor as +1-+100 with respect to pleasantness; if theyconsidered the odor unpleasant (“they did not wish to smell the odoragain”) they graded the odor as −1-−100 with respect to unpleasantness;if they did not consider the odor either pleasant or unpleasant theygraded the odor as neutral or 0. Results were obtained by calculatingthe arithmetical sum of each correct recognition response for each odorwith respect to its pleasantness, unpleasantness or neutrality.Arithmetic M±SEM were obtained for each treatment group for each odorpresented.

Independently, patients were also required to grade their ability tosmell daily on a scale from 0-100, with 0 reflecting no overall smellfunction over a 24 hour period, 100 reflecting normal overall smellfunction over this period and numbers between 0-100 reflecting theirestimation of their overall ability to smell odors over this period.

Based upon values for the DT, RT and ME tests, patients were classifiedwith respect to severity of smell loss into the four types [(1, 2)(Table I)]. Anosmia is the complete loss of smell. Patients with anosmiahave DT, RT and ME test values of zero, since they cannot detect,recognize nor grade the intensity of any odor including an absoluteconcentration of any odorant (Table I). No patients with anosmia werepresent in the study due to the relative rarity of this condition (1).Patients with Type I hyposmia (96 patients) could detect some odors butcould not recognize any odor correctly; thus, DTs for some odors werepresent, but RTs and MEs for all odors were zero since they couldneither recognize correctly nor thereby grade correctly intensity of anyodor (Table I). Patients with Type II hyposmia (208 patients) coulddetect and recognize some odors, but at levels greater than normal; thusDTs and RTs were present, but elevated above normal and MEs were presentbut at levels lower than normal (Table I). Patients with Type IIIhyposmia (8 patients) could detect and recognize all odors at normallevels (i.e., normal DT and RT), but ME values for one or more odorswere significantly decreased below normal (Table I). Severity of smellloss graded from most to least severe loss was typed asanosmia >hyposmia Type I>Type II>Type III and verified by demonstratingthat as smell loss severity increased levels of nasal mucus cAMP andcGMP decreased (32, 34).

TABLE I: CLASSIFICATION OF SMELL LOSS DETECTION RECOGNITION MAGNITUDETHRESHOLD THRESHOLD ESTIMATION DT in M/L RT in M/L MEAN ME in %NORMALS + +* ≥48 PATIENTS ANOSMIA∞ 0 0 0 HYPOSMIA TYPE I ± 0 0 TYPE II ±±* <48 + Normal (≤10^(−5M) for all odorants) +*Normal (≤10^(−2M) for allodorants) 0 Absent response (∞) ± Present but < normal (>10^(−5M) < ∞for all odorants) ±*Present but < normal (>10^(−2M) < ∞ for allodorants) ∞Inability to detect, recognize or judge intensity of anabsolute concentration of odorant

After determination of hyposmia, patients were treated in an open label,fixed design, controlled open trial. Patients were given an oralextended release theophylline in divided daily doses taken in the middleof breakfast and lunch. All patients were initially given 200 mg oftheophylline; changes in this dose were made based upon subjectiveresponses to therapy. If at a subsequent return visit, patients reported≥5% subjective improvement in overall smell function, they continued onthis same dose of theophylline and were reevaluated after four to sixmonths of continued treatment. Results from any subsequent return fromthese improved patients were not included in any subsequent data report.All subsequent comparisons between treated and untreated patients weremade only between those patients continuing in the study compared totheir own measurements obtained in the untreated state. If patientsreported <5% subjective improvement, their theophylline dose wasincreased by 200 mg daily and they were scheduled for retesting at thestudy site after two to four months at this new dosage. For patientsthat reported <5% improvement, this sequence continued until patientsreached 800 mg of theophylline, at which time the study was consideredcompleted.

Patients were initially divided into two groups based upon proximity tothe study site. One group, consisting of local patients (212 patients),returned for reevaluation after two-four months of treatment. The othergroup, consisting of distant patients (100 patients), called the studysite at two to four month intervals and visited the study site after sixto 10 months of treatment. Some of the distant patients returned aftertreatment on 200 mg, 400 mg, 600 mg of theophylline and these resultswere included with those of local patients at each dose level (see FIG.1 for patient details).

Local Patients—First Return (200 mg Theophylline)

Patients returned for reevaluation after two to four months oftreatment. Measurements of blood serum theophylline were measured by afluorescence polarization assay (Abbott, Chicago, Ill.). At this time,subjective changes in smell function were measured independently(compared to patient's memory of previous normal smell functionmonitored by use of the patients' daily measurements) using the scalefrom −100-0-+100 previously described (vs). After independentlyrecording their subjective responses, smell function was measured forall four odors with DT, RT, ME and H determined without recourse toprior measurements.

Local Patients—Second Return (400 mg Theophylline)

Patients returned after four to eight months of treatment and consistedof patients who previously reported <5% improvement on 200 mg oftheophylline but also some distant patients who returned on this dose.Blood theophylline was measured as before. All patients independentlyreported subjective overall changes in smell function as donepreviously. At this visit, smell function was measured as before todetermine DT, RT, ME and H for all four odors without recourse to priormeasurements.

Local Patients—Third Return (600 mg Theophylline)

Patients returned after four-10 months of treatment and included bothpatients who reported <5% improvement on 400 mg theophylline and alsosome of the distant patient group. Blood theophylline was obtained asbefore. Patients independently reported subjective changes in overallsmell function and measurements of DT, RT, ME and H were obtained asbefore for all four odors without recourse to prior measurements.

Local Patients—Fourth Return (800 mg Theophylline)

Patients returned after four-eight months of treatment and includedpatients who reported <5% improvement on 600 mg theophylline and alsosome of the distant patient group. Blood theophylline was obtained asbefore. Patients independently reported subjective changes in overallsmell function and measurements of DT, RT, ME and H were obtained asbefore for all four odors without recourse to prior measurements.

Distant Patients—First Call in (200 mg Theophylline)

Subjective overall changes in smell acuity were reported by telephoneand by FAX or email at two-four month intervals after treatment wasinitiated using the standardized form in which they measured dailychanges in smell acuity using the −100-0-+100 scale as noted above (vs).Using the same criteria noted above, if at their initial two-four monthcall-in they reported improvement in smell acuity of ≥5%, they continuedat 200 mg and returned to the study site at four-six months on thistreatment dose. If they reported improvement in smell acuity of <5%their dose of theophylline was increased to 400 mg for an additionaltwo-four months and they then called the study site at the end of thisperiod.

Distant Patients—Second Call in (400 mg Theophylline)

At this time, if patients reported ≥5% improvement on 400 mg theycontinued on this dose and returned to the study site after four-sixmonths on this treatment dose. If they reported <5% improvement in smellfunction their theophylline dose was increased to 600 mg and they thencalled the study site after four-six months on this treatment dose.

Distant Patients—Third Call in (600 mg Theophylline)

At this time, if patients reported ≥5% improvement they were continuedon this treatment dose and were requested to return to the study site infour-six months. If they reported <5% improvement in smell function theywere requested to return to the study site as soon as possible toreevaluate their smell function. At this return, blood theophyllinelevels were measured. Smell function was measured using the subjectiveand psychophysical techniques (DT, RT, ME, H) previously described.

Distant Patients—Fourth Call in (800 mg Theophylline)

At this time, if patients reported either ≥5% improvement or <5%improvement, they were requested to return to the study site as soon aspossible to reevaluate their smell function. At this return, bloodtheophylline levels were measured and smell function were measured usingboth subjective and psychophysical techniques with results as shown onTable VII and discussed previously.

Since data for all measurements from each patient group (local ordistant) on each dose level were combined mean and SEM for eachmeasurement (DT, RT, ME, H) of smell function for each treatment groupwere calculated on this basis. Differences in measurements betweenbefore treatment and each treatment level were calculated andsignificance of differences estimated using Student t tests (valuesp<0.05 considered significant). Some patient responses were alsoanalyzed by use of non-parametric statistics (sign test) and theSpearman rank correlation technique; r<0.05 was considered significantrelated to smell loss type and treatment response. Since each patientserved as his/her own control with respect to before and aftertreatment, paired t tests were also calculated; t values <0.05 wereconsidered significant. These values were not included in the resultspresented.

Results were previously obtained in 150 normal subjects for each ofthese measurements (1, 53-55) and are reported here for comparison.

Results

Comparison of Smell Function in Normal Subjects and in UntreatedPatients with Hyposmia

Results of each measurement (DT, RT, ME and H) in untreated patientsindicated significant impairment of smell function compared to normalsubjects (Table II, FIG. 2A-B). For DT and RT, patient responses weresignificantly higher (less sensitive) than in normals (Table II), MEresponses were significantly lower (less sensitive) than in normals(FIG. 2A-B). H responses were significantly lower (less pleasant) foramyl acetate and nitrobenzene (odors usually considered pleasant) andsignificantly higher for pyridine and thiophene (less unpleasant—closerto zero for odors usually considered unpleasant) (FIG. 2A-B). Analysisof H data indicate that for normal subjects H values for pyridine andthiophene (unpleasant responses) are similar to or slightly higher thanME values, whereas H values for nitrobenzene and amyl acetate (pleasantresponses) are similar or slightly lower. Ratios of H:ME in normals forpyridine and thiophene are 1.09 and 1.05 whereas for nitrobenzene andamyl acetate they are 0.94 and 0.96. For untreated patients (patientswith Type II and III hyposmia only) these ratios are quite different.For pyridine and thiophene, these ratios are 0.87 and 0.81 whereas fornitrobenzene and amyl acetate they are 0.81 and 0.10. In part, Hdecreases for nitrobenzene and amyl acetate are related to decreasedpatient acuity; however, the major discrepancy is that about 50% ofpatients with hyposmia also exhibit dysosmia (1, 2) in which odorsusually considered pleasant are considered unpleasant (e.g., banana-oilodor may be considered putrid) and even unpleasant odors may beconsidered pleasant (e.g., pyridine may be considered flowery). Thesedistortions in patients comprise a bimodal response for H values (somepatients reporting a normal hedonic response related to appropriatepleasantness and unpleasantness of the perceived odor whereas othersreporting a distortion) which reduces the overall arithmetic meanobtained for H for each odor.

TABLE II DIFFERENCES IN SMELL FUNCTION BETWEEN STUDY PATIENTS BEFORETREATMENT AND NORMAL CONTROLS SMELL FUNCTION\ PATIENTS (312) NORMALS(155) ODOR PYRD NO₂B THIO AA PYRD NO₂B THIO AA DT    8.5 ± 0.2*^(, a) 9.0 ± 0.2^(a)  8.5 ± 0.2^(a) 8.9 ± 0.2^(a) 4.2 ± 0.1 3.9 ± 0.1 3.6 ±0.1 3.7 ± 0.1 RT 10.2 ± 0.1^(a) 10.5 ± 0.2^(a) 10.2 ± 0.2^(a) 10.7 ±0.01^(a) 7.2 ± 0.1 5.7 ± 0.1 7.1 ± 0.1 6.8 ± 0.1 ME 23 ± 2^(a) 12 ±1^(a) 16 ± 2^(a) 10 ± 1^(a)  64 ± 3  51 ± 4  66 ± 4  51 ± 4  H −20 ±2^(a)   4 ± 1^(a) −13 ± 2^(a)  1 ± 1^(a) −74 ± 3  48 ± 5  −80 ± 2  54 ±5  ( ) Patient number *Mean ± SEM DT, detection threshold, in bottleunits [BU ( )] RT, recognition threshold, in bottle units [BU ( )] ME,magnitude estimation, in % ( ) H, hedonic estimation, in % (+, pleasant,−, unpleasant, 0, neutral) PYRD, pyridine; NO₂B, nitrobenzene; THIO,thiophene; AA, amyl acetate With respect to normals ^(a)p < 0.001

Comparison of Smell Function in Patients Classified by Degree of SmellLoss

Categorized by smell loss type, results of each measurement indicatethat before treatment, patients exhibited a consistent pattern ofabnormality associated with their loss type such that Type Ihyposmia>Type II>Type III (Table III). H:ME ratios were closer to thoseof normals in patients with Type III compared to those with Type IIhyposmia, as would be expected due to the lesser degree of abnormalityin the former patients (Table III).

TABLE III COMPARISON OF SMELL FUNCTION BETWEEN UNTREATED PATIENTS WITHTYPES I, II AND III HYPOSMIA I (96) II (208) PYRD NO₂B THIO AA PRYD NO₂BTHIO DT 9.8 ± 0.1* 11.2 ± 0.2 10.9 ± 0.2 11.7 ± 0.2 8.8 ± 0.2  8.3 ±0.2^(a) 8.0 ± 0.2^(a) RT 11.7 ± 0.1  11.8 ± 0.1 11.8 ± 0.1 11.8 ± 0.1 9.7 ± 0.1^(a) 10.1 ± 0.2^(a) 9.7 ± 0.2^(a) ME 0 0 0 0 29 ± 2^(a ) 16 ±1^(a) 21 ± 2^(a)  H 0 0 0 0 −25 ± 2^(a )  5 ± 1^(a) −16 ± 2^(a)  II(208) III (8) AA PRYD NO₂B THIO AA DT  8.2 ± 0.2^(a) 4.0 ± 0.5 3.4 ±0.6^(a, a1) 3.3 ± 0.8^(a, a1)  2.5 ± 0.5^(a, a1) RT 10.4 ± 0.2^(a) 5.6 ±0.8 4.0 ± 0.9^(a, a1) 3.9 ± 0.8^(a, a1) 5.3 ± 1.0  ME 13 ± 1^(a)  42 ±4^(a, a1) 34 ± 4^(a, a1)  34 ± 10^(a )  40 ± 2^(a, a1) H  1 ± 1 −27 ±14^(a)  12 ± 7^(a )   −46 ± 13^(a   )  34 ± 6^(a, a1) ( ) Patient number*Mean ± SEM DT, detection threshold, in BU RT, recognition threshold, inBU ME, magnitude estimation, in % H, hedonic estimation, in % PYRD,pyridine; NO₂B, nitrobenzene; THIO, thiophene; AA, amyl acetate Withrespect to Type I ^(a)p < 0.001 With respect to Type II ^(a1)p < 0.001

Changes in Smell Function in all Patients Treated with Theophylline, 200mg Daily

Changes in smell function in 199 patients with hyposmia after treatmentwith 200 mg daily of theophylline for two-six months are shown in TableIV and in FIG. 3A-B. These patients constituted mainly local patientsminus 15 of the original 312 who did not return after treatment wasinitiated. Of the 15 non-returning patients, seven reported >5%improvement (46.7%) and one (14.3%) reported a return to normal; eightreported improvement of <5%. With returning patients, 34 patients(17.1%) improved ≥5% (and were not included in subsequent studies).Among this group nine (26.5%) considered their smell function hadreturned to normal. Two hundred sixty-three patients improved <5% (160local and 103 distant patients); among this group 36 patients did notcontinue in the study.

Among the 199 patients (both improved and not improved) who returned on200 mg, DT and RT for all odors decreased (improved) significantly(Table IV). ME for pyridine and amyl acetate also increased (improved)significantly (FIG. 3A-B). H values for pyridine and thiophene decreasedsignificantly [i.e., odors pyridine and thiophene were recognized asmore unpleasant (FIG. 3A-B)] and H values for nitrobenzene increasedsignificantly [i.e., the odor of nitrobenzene was recognized as morepleasant (FIG. 3A)]. While not statistically significant, H values foramyl acetate increased 50% (perceived as more pleasant with improvedodor recognition). On treatment, H:ME ratios did not changesignificantly since both ME and H values changed to similar degrees. Nodifferences in results with respect to age or gender of these patientswere apparent. At subsequent visits over the next six-36 months withcontinued treatment on this dose, the 34 patients with initiallyimproved smell function maintained their increased acuity or improvedfurther. Mean serum theophylline on this treatment was 4.0±0.2

TABLE IV CHANGES IN SMELL FUNCTION FOLLOWING TREATMENT WITH ORALTHEOPHYLLINE, 200 MG DAILY SMELL FUNCTION\ Before Treatment (199) AfterTreatment (199) ODOR PYRD NO₂B THIO AA PYRD NO₂B THIO AA DT  8.5 ± 0.2* 9.0 ± 0.2  8.5 ± 0.2  8.9 ± 0.2 7.6 ± 0.2^(a) 7.8 ± 0.2^(d) 7.5 ±0.2^(c) 7.9 ± 0.2^(c) RT 10.2 ± 0.1 10.5 ± 0.2 10.2 ± 0.2 10.7 ± 0.1 9.0± 0.2^(a) 9.5 ± 0.2^(a) 9.1 ± 0.2^(c) 9.9 ± 0.2^(c) ME 23 ± 2 12 ± 1 16± 2 10 ± 1 28 ± 2^(a)  17 ± 2  21 ± 2  15 ± 2^(d)  ( ) Patient number*Mean ± SEM DT, detection threshold, in BU RT, recognition threshold, inBU ME, magnitude estimation, in % H, hedonic estimation, in % PYRD,pyridine; NO₂B, nitrobenzene; THIO, thiophene; AA, amyl acetate Withrespect to before treatment ^(a)p < 0.001 b p < 0.005 ^(c)p < 0.01 ^(d)p< 0.05

Changes in Smell Function in Patients with Hyposmia Treated withTheophylline 400 mg Daily

One hundred twenty patients (97 from the 165 who returned on <5% on 200mg and 23 who returned for the first time on 400 mg) were reevaluatedafter taking 400 mg of theophylline for two-six months (Table IV). Onthis treatment dose, 35 patients (29.2%) improved ≥5% (and were notincluded in subsequent studies). Among this group, three (8.6%)considered their smell function had returned to normal. One hundredninety-two patients improved <5% (96 local and 96 distant patients).Twenty-five patients of these 192 did not continue in the study. On thisdose, DT and RT for all odors decreased (improved) significantly and MEfor all odors increased (improved) significantly (Table V) (bothimproved and unimproved patients included). Hedonic values alsoincreased significantly for pyridine and thiophene [i.e., odors ofpyridine and thiophene were recognized as more unpleasant (FIG. 2A-B)].While not statistically significant, H values for nitrobenzene increased50% (perceived as more pleasant); H values for thiophene decreased 40%(perceived as more unpleasant). Overall, H:ME ratios did not changesignificantly on treatment. No differences in results with respect toage or gender of these patients were apparent. Among patients whoexhibited improvement at this dose, on subsequent visits over six-36months, their improvement persisted. Mean serum theophylline on thistreatment dose was 7.4±0.4

TABLE V CHANGES IN SMELL FUNCTION FOLLOWING TREATMENT WITH ORALTHEOPHYLLINE, 400 MG DAILY SMELL FUNCTION\ Before Treatment (120) AfterTreatment (120) ODOR PYRD NO₂B THIO AA PYRD NO₂B THIO AA DT  8.4 ± 0.2* 9.0 ± 0.3  8.6 ± 0.3  8.9 ± 0.3 7.0 ± 0.3^(c) 7.2 ± 0.3^(c) 6.8 ±0.4^(c) 7.1 ± 0.3^(b) RT 10.3 ± 0.2 10.5 ± 0.2 10.5 ± 0.2 10.6 ± 0.2 8.5± 0.3^(a) 8.9 ± 0.3^(b) 8.4 ± 0.3^(a) 9.3 ± 0.3^(b) ME 21 ± 2 12 ± 2 14± 2 10 ± 1 33 ± 3^(a)  23 ± 2^(a)  26 ± 2^(c)  18 ± 2^(c)  H −19 ± 2   4± 1 −12 ± 2   2 ± 1 −26 ± 3^(b)  8 ± 3^(a) −19 ± 2^(d)  3 ± 2  ( )Patient number *Mean ± SEM DT, detection threshold, in BET RT,recognition threshold, in BU ME, magnitude estimation, in % H, hedonicestimation, in % PYRD, pyridine; NO₂B, nitrobenzene; THIO, thiophene;AA, amyl acetate With respect to before treatment ^(a)p < 0.001 ^(b)p <0.005 ^(c)p < 0.01 ^(d)p < 0.05

Change in Smell Function in Patients with Hyposmia Treated withTheophylline, 600 mg Daily

Changes in smell function in 160 patients after treatment with 600 mgdaily for two-12 months are shown on Table VI. This patient numberincludes 77 distant patients who returned on this theophylline dose aswell as 83 local patients who improved <5% on 400 mg. Among this group,66 (41.2%) improved ≥5% and 17 (10.6%) considered their smell functionhad returned to normal. On subsequent visits, over the next six-36months, improvement on this dose either persisted or improved further.One hundred thirty-seven patients improved <5% and 73 did not continue.On this dose, DT and RT for all odors decreased (improved) significantlyand ME for all odors increased (improved) significantly (Table VI) (bothimproved and unimproved patients included). H values did not changesignificantly for any odor, although pyridine and thiophene wererecognized as more unpleasant (FIG. 3A-B) and nitrobenzene and amylacetate were recognized as more pleasant (FIG. 3A-B). Overall, H:MEratios did not change significantly. Again, as noted on the previoustheophylline doses, no differences were apparent with respect to age orgender of these patients. Mean serum theophylline on this dose was9.4±0.38 mg/dl.

TABLE VI CHANGES IN SMELL FUNCTION FOLLOWING TREATMENT WITH ORALTHEOPHYLLINE, 600 MG DAILY SMELL FUNCTION\ Before Treatment (160) AfterTreatment (160) ODOR PYRD NO₂B THIO AA PYRD NO₂B THIO AA DT  8.4 ± 0.2* 9.2 ± 0.2  8.8 ± 0.2 9.3 ± 0.2 7.4 ± 0.2^(c) 7.8 ± 0.3^(a) 7.1 ±0.3^(a) 7.8 ± 0.3^(a) RT 10.3 ± 0.2 10.5 ± 0.2 10.3 ± 0.2 10.8 ± 0.2 9.4 ± 0.2^(c) 9.1 ± 0.2^(c) 9.0 ± 0.3^(c) 9.6 ± 0.2^(a) ME 19 ± 2 11 ± 113 ± 2 8 ± 1 26 ± 2^(d)  18 ± 2^(d)  21 ± 2^(d)  15 ± 2^(c)  H −16 ± 2  3 ± 1 −9 ± 2 2 ± 1 −19 ± 2   8 ± 2^(d) −14 ± 2   4 ± 1  ( ) Patientnumber *Mean ± SEM DT, detection threshold, in BET RT, recognitionthreshold, in BU ME, magnitude estimation, in % H, hedonic estimation,in % PYRD, pyridine; NO₂B, nitrobenzene; THIO, thiophene; AA, amylacetate With respect to before treatment ^(a)p < 0.001 b p < 0.005 ^(c)p< 0.01 ^(d)p < 0.05

Change in Smell Function in Patients with Hyposmia Treated withTheophylline, 800 mg Daily

Changes in smell function in 28 patients after treatment with 800 mgdaily for two-12 months are shown in Table VII. Among this group, 15(53.6%) improved ≥5% and three (33%) considered their smell function hadreturned to normal. On subsequent visits over the next two-six months,improvement on this dose persisted or improved further. Thirteenpatients improved <5%. At this dose, the study terminated. DT and RT forall odors increased on treatment, but were statistically significantonly for DT for nitrobenzene, RT for nitrobenzene and amyl acetate. MEfor all odors increased, but was significant only for amyl acetate. Hfor both pyridine and thiophene decreased 55% and 37%, respectively(became more unpleasant) and H for nitrobenzene and amyl acetateincreased about 50% (became more pleasant). When analyzed by paired ttest (data not shown) DT, RT and ME for all odors increasedsignificantly, H for pyridine and thiophene decreased significantly andH for nitrobenzene and amyl acetate increased significantly. Mean serumtheophylline on this dose was 11.2±0.8 mg/dl.

As drug doses increased mean DT and RT for most odors decreased (acuityincreased) whereas, mean ME for most odors increased from 200 mg to 400mg and then remained relatively constant. H also decreased from 200 mgto 400 mg (increased unpleasantness) for pyridine and thiophene and thenremained relatively constant as doses increased; H for nitrobenzene andamyl acetate (increased pleasantness) increased in a similar manner.

TABLE VII CHANGES IN SMELL FUNCTION FOLLOWING TREATMENT WITH ORALTHEOPHYLLINE, 800 MG DAILY SMELL FUNCTION\ Before Treatment (28) AfterTreatment (28) ODOR PYRD NO₂B THIO AA PYRD NO₂B THIO AA DT  7.9 ± 0.6* 9.3 ± 0.7  8.9 ± 0.6  8.8 ± 0.7 6.9 ± 0.6 7.0 ± 0.8^(d) 7.2 ± 0.7 7.2 ±0.8 RT 10.6 ± 0.3 10.1 ± 0.6 10.2 ± 0.5 10.9 ± 0.3  8.5 ± 0.6^(c) 7.8 ±0.8^(d) 9.6 ± 0.6  9.0 ± 0.5^(b) ME 18 ± 4 11 ± 4 14 ± 4 10 ± 4 29 ± 5 25 ± 6   20 ± 4  24 ± 2^(a ) H −12 ± 4   6 ± 2 −9 ± 4  4 ± 4 −22 ± 6  13± 7   −15 ± 4  8 ± 5 ( ) Patient number *Mean ± SEM DT, detectionthreshold, in BU RT, recognition threshold, in BU ME, magnitudeestimation, in % H, hedonic estimation, in % PYRD, pyridine; NO₂B,nitrobenzene; THIO, thiophene; AA, amyl acetate With respect to beforetreatment ^(a)p < 0.001 ^(b)p < 0.005 ^(c)p < 0.01 ^(d)p < 0.05

Changes in Smell Function and after Treatment with Theophylline inPatients Classified by Hyposmia Type

Type I Hyposmia Treatment. After treatment with either 200 mg, 400 mg,600 mg or 800 mg, there were significant decreases in DT and RT,increases in ME and changes in H for specific odors consistent withimprovement in smell function (Table VIII). Of the 96 patients with TypeI hyposmia in the study, 32 (33.3%) reported >5% improvement and 5(15.6%) reported their smell function had returned to normal.

TABLE VIII CHANGES IN SMELL FUNCTION IN TYPE I HYPOSMIA PATIENTSFOLLOWING TREATMENT SMELL FUNCTION\ Before Treatment (96) ODOR PYRD NO₂BTHIO AA PYRD NO₂B THIO AA After Treatment 200 mg (57) DT 9.8 ± 0.1* 11.2± 0.2 10.9 ± 0.2 11.2 ± 0.2  9.5 ± 0.2 10.3 ± 0.3^(a)  10.1 ± 0.3^(d) 10.5 ± 0.3^(d)  RT 11.7 ± 0.1  11.8 ± 0.1 11.8 ± 0.1 11.7 ± 0.1 11.0 ±0.2^(a) 11.2 ± 0.2^(d)  11.4 ± 0.2  11.3 ± 0.2  ME 0 0 0 0 11 ± 2^(a) 5± 2^(a) 6 ± 2^(a) 6 ± 2^(a) H 0 0 0 0 −8 ± 2^(a) 4 ± 2^(a) −3 ± 2^(a)  4± 2^(a) After Treatment 400 mg (33) DT  8.8 ± 0.5 9.8 ± 0.5^(a) 9.8 ±0.5^(a) 10.1 ± 0.5^(a)  RT 10.6 ± 0.4  10.9 ± 0.4^(e)  10.8 ± 0.4^(d) 11.2 ± 0.3  ME 12 ± 3^(a) 8 ± 3^(a) 7 ± 2^(a) 7 ± 3^(a) H −7 ± 3^(a) 3 ±3^(a) −5 ± 2^(a)  5 ± 3^(a) After Treatment 600 mg (51) DT  8.9 ±0.3^(b) 9.9 ± 0.4^(c) 9.4 ± 0.4^(a) 10.1 ± 0.3^(a)  RT 10.9 ± 0.3  10.8± 0.3^(e)  10.9 ± 0.3^(b)  11.1 ± 0.2^(c)  ME 11 ± 3^(a) 6 ± 2^(a) 7 ±2^(a) 5 ± 2^(a) H −8 ± 2^(a) 2 ± 2  −3 ± 2^(a)  −0.4 ± 1    AfterTreatment 800 mg (12) DT  9.6 ± 0.8 10.1 ± 0.8  10.0 ± 0.7  10.6 ± 0.5 RT 10.3 ± 1.0  11.3 ± 0.7  11.6 ± 0.2  11.6 ± 0.4  ME 10 ± 4^(a) 4 ±3^(a) 4 ± 2^(a) 5 ± 3^(a) R −11 ± 5^(a)  2 ± 2^(a) −4 ± 2^(a)  −1 ± 4  ( ) Patient number; *Mean ± SEM DT, detection threshold, in BU RT,recognition threshold, in BU ME, magnitude estimation, in % H, hedonicvalue, in % PYRD, pyridine; NO₂B, nitrobenzene; THIO, thiophene; AA,amyl acetate Compared to before treatment ^(a)p < 0.001 ^(b)p < 0.005^(c)p < 0.01 ^(d)p < 0.05

Type II Hyposmia Treatment. After treatment with either 200 mg, 400 mg,600 mg or 800 mg, there were significant decreases in DT and RT,increases in ME and changes in H for specific odor consistent withimprovement in smell function (Table IX). Of the 208 patients with TypeII hyposmia in the study, 129 (62%) reported >5% improvement and 26(20.2%) reported their smell function had returned to normal.

TABLE IX CHANGES IN SMELL FUNCTION IN TYPE II HYPOSMIA PATIENTSFOLLOWING TREATMENT SMELL FUNCTION\ Before Treatment (208) ODOR PYRDNO₂B THIO AA PYRD NO₂B THIO AA After Treatment 200 mg (138) DT 8.8 ± 0.2 8.3 ± 0.2 8.0 ± 0.2  8.2 ± 0.2 6.9 ± 0.2  6.9 ± 0.3^(a) 6.5 ± 0.3  7.0± 0.3  RT 9.7 ± 0.1 10.1 ± 0.2 9.7 ± 0.2 10.4 ± 0.2 8.2 ± 0.2^(a) 8.8 ±0.3  8.2 ± 0.3^(a) 9.3 ± 0.2  ME 29 ± 2  16 ± 1 21 ± 2  13 ± 1 35 ± 2 22 ± 2  27 ± 2  19 ± 2  H −25 ± 2   5 ± 1 −16 ± 2   1 ± 1 −28 ± 2   7 ±2  −19 ± 2   3 ± 2  After Treatment 400 mg (85) DT 6.3 ± 0.3^(a) 6.3 ±0.4^(a) 5.7 ± 0.4  6.1 ± 0.4^(a) RT 7.7 ± 0.3^(a) 8.2 ± 0.4^(a) 7.6 ±0.4^(a) 8.8 ± 0.4^(a) ME 41 ± 3^(a)  27 ± 3^(a)  31 ± 3  21 ± 3  H −34 ±3   11 ± 3  −23 ± 3   0.3 ± 2.8  After Treatment 600 mg (105) DT 6.7 ±0.3^(a) 6.8 ± 0.3^(a) 6.1 ± 0.3^(a) 6.8 ± 0.3^(a) RT 8.9 ± 0.3  8.4 ±0.3  8.1 ± 0.3^(a) 8.9 ± 0.3^(a) ME 33 ± 2  24 ± 2  27 ± 2  19 ± 2  H−25 ± 3   11 ± 2  −20 ± 2   7 ± 2  After Treatment 800 mg (16) DT 4.7 ±0.7^(a) 4.6 ± 0.9^(a) 4.9 ± 0.7^(a) 4.6 ± 0.7^(a) RT 7.1 ± 0.7^(a) 5.1 ±0.9^(a) 8.0 ± 1.0  7.1 ± 0.6^(a) ME 43 ± 5^(e)  42 ± 8^(b)  33 ± 6  38 ±6^(e)  H −31 ± 9   22 ± 11  −24 ± 8   13 ± 9  ( ) Patient number DT,detection threshold, in BU RT, recognition threshold, in BU ME,magnitude estimation, in % H, hedonic value, in % PYRD, pyridine; NO₂B,nitrobenzene; THIO, thiophene; AA, amyl acetate Compared to beforetreatment ^(a)<0.001 ^(b)p < 0.005 ^(c)p < 0.02

Type III Hyposmia Treatment. After treatment with either 200 mg, 400 mgor 600 mg, there were no significant changes in DT or RT, since thesevalues were not significantly different from normal before treatment.After treatment, there were changes in ME and H, but values werevariable due to the small number of patients in each treatment series(Table X). Of the eight patients with Type III hyposmia in the study, 5(62.5%) reported >5% improvement and 3 (60%) reported their smellfunction had returned to normal (see Table X).

TABLE X CHANGES IN SMELL FUNCTION IN TYPE III HYPOSMIA PATIENTSFOLLOWING TREATMENT SMELL FUNCTION\ Before Treatment (8) ODOR PYRD NO₂BTHIO AA PYRD NO₂B THIO AA After Treatment 200 mg (4) DT 4.0 ± 0.5 3.4 ±0.6 3.3 ± 0.8 2.5 ± 0.5 4.0 ± 0.6 3.8 ± 0.7 3.2 ± 0.8 2.8 ± 0.5 RT 5.6 ±0.8 4.0 ± 0.9 3.9 ± 0.8 5.3 ± 1.0 5.2 ± 0.5 5.0 ± 0.6 3.8 ± 0.6 5.2 ±1.6 ME 42 ± 4  34 ± 4  34 ± 10 40 ± 2  45 ± 11 33 ± 8  47 ± 15 32 ± 10 H−27 ± 14  12 ± 7  −46 ± 13  34 ± 8  −40 ± 13  13 ± 12 −40 ± 18  14 ± 13After Treatment 400 mg (2) DT 5.0 2.5 1  2 RT 5.0 4.0 1  3 ME 45   79  99  75 H −40    −62    −99  75 After Treatment 600 mg (4) DT 3.8 ± 0.93.8 ± 0.9 2.0 ± 1.0 2.5 ± 1.0 RT 5.5 ± 0.9 4.5 ± 1.3 3.8 ± 0.9 4.8 ± 1.7ME 41 ± 15 33 ± 13 42 ± 20 31 ± 17 H −26 ± 22  12 ± 16 −35 ± 24  28 ± 19( ) Patient number DT, detection threshold, in BU RT, recognitionthreshold, in BU ME, magnitude estimation, in % H, hedonic value, in %PYRD, pyridine; NO₂B, nitrobenzene; THIO, thiophene; AA, amyl acetate

Discussion

These results indicate that 157 of the 312 patients in the study (50.3%)were responsive to treatment with theophylline. Of these, 34 (21.7%)considered their smell function returned to normal levels. Overall,10.9% of all patients in the study considered their smell function hadreturned to normal. Improvement in smell function, once occurred,persisted and sometimes continued to improve as long as treatmentcontinued.

Initial studies were made at two-six month intervals after druginitiation. Subjective responses indicated that return of function wasboth time and dose related; patients reported little or no improvementbefore four-six weeks of treatment and reported greater improvement asdoses increased to 400 mg and especially to 600 mg and 800 mg.

Patients with smell loss exhibited varying degrees of loss prior totreatment, however, improvement was noted in patients regardless ofdegree of loss. Patients with lesser degrees of smell loss (Type II andIII hyposmia) exhibited more improvement with more patients reporting areturn to normal smell function on treatment than those with a moresevere degree of loss (Type I hyposmia). Indeed, in terms of percentpatients reporting a return to normal function, twice as many patientswith Type II and III hyposmia compared to Type I reported a return tonormal function. These results are useful since smell loss degree isrelated to severity of biochemical changes (35) responsible for theloss. This result lends credence to the smell loss classificationpreviously devised (1, 2, 56) and alerts physicians treating thesepatients that patients with a greater degree of smell loss requiregreater care and diligence with respect to successful treatmentresponses. Among patients who did not improve (those who responded <5%improvement), increasing the dose of theophylline further to 800 mgimproved smell function further. In another group of patients whoimproved <5%, the addition of another PDE inhibitor (i.e., cilostazol)to their dose of 600 mg or 800 mg of theophylline improved smellfunction in an additional 15% of patients.

When analyzed with respect to changes in objective psychophysicalmeasurements of smell function, mean values for DT and RT improvedsignificantly for all odors for each theophylline dose, whether or notsubjective improvement occurred (Tables IV, V, VI). These resultsindicate that treatment with theophylline improved standardizedthreshold measurements of sensory function, albeit, not enough to beperceived by the patients as significant. While mean DT and RT for allodors improved on treatment, none of these means reached the level ofimprovement exhibited by normal subjects (cf, Tables III, IV, V, VI).

When analyzed with respect to ME on 200 mg theophylline (Table IV), onlyresponses to pyridine were significantly greater than prior totreatment. ME for each odor, however, increased with the total meanincrease (5.1%) consistent with the overall reported subjectiveimprovement among the successfully treated patients. On 400 mgtheophylline (Table V), ME for each odor increased significantly with atotal mean increase of 10.4%. On 600 mg theophylline (Table VI), ME foreach odor also increased significantly with a total mean increase of6.9%. These results are probably more consistent with patient subjectivechanges, since most patients are less focused on whether or not they candetect or recognize weak (threshold) concentrations of odors. Rather,they are more concerned about the intensity at which odors areperceived. These results are consistent with the subjective responses ofthe patients.

In general, as noted before, ME and H values in normal subjects aresimilar, since subjects usually equate pleasantness or unpleasantness ofany odor with its intensity, be it pleasant or unpleasant (Table V). Forexample, pyridine odor of 50% intensity is generally considered as 50%unpleasant whereas amyl acetate odor of 50% intensity is generallyconsidered as 50% pleasant. Patients with hyposmia, however, may notonly manifest decreased sensory acuity, but also sensory distortions.This phenomena was observed upon comparison of ME and H values betweenuntreated patients and normals (Table II). The disparate ratios of H:MEreflected among the patients not only manifest decreased acuity (lowerME) but also the bimodal distribution of H values (as discussedpreviously). This bimodal distribution becomes more apparent for Hvalues as patients recovered their sensory acuity (manifested byincreased DT, RT and ME) since there was an even greater divergence ofpleasantness-unpleasantness among all odors presented than in normals.Thus, H values among some patients in whom sensory acuity increased,pleasant odors (e.g., nitrobenzene—bitter almond or marzipan-like oramyl acetate—banana-like) were considered putrid because the distortedaspect of these odors also increased. This type of change may not be asreadily apparent with respect to changes in more unpleasant odors (e.g.,pyridine, thiophene), but may also occur with these unpleasant odorsconsidered sweet or fruity as part of the distortion.

On 200 mg theophylline, ME values for pyridine and thiophene increased6% and 5%, respectively, whereas H values decreased (became lessunpleasant) 2%, respectively; ME values for nitrobenzene and amylacetate increased 5%, respectively, whereas H values increased (becamemore pleasant) only by 2% (Table IV). This effect is better appreciatedon 400 mg theophylline with ME increases for pyridine and thiophene at12%, respectively, whereas increases in H values were 9% and 7%,respectively; ME for nitrobenzene and amyl acetate increased 11% and 8%,respectively, whereas H values decreased (became more pleasant) only 4%and 1%, respectively (Table V). Similar results hold for 600 mgtheophylline also (Table VI).

Differences with respect to subjective responses and changes measured inDT and RT before and after treatment may reflect differences in howpatients considered their overall improvement on treatment. A responseof ≥5% was chosen to indicate improvement in smell function ontreatment. This apparently small response number may in actuality be aconservative estimate of return of smell function since this number is acomposite of all odors which the patient considered improved ontreatment. Thus, responses to some strong odors (e.g., gasoline, bleach,ammonia, etc.) may have been considered improved by a great deal, butthese responses may have been tempered by the patients' responses toweaker odors (e.g., flowers, perfume, shampoo, etc.) in which noimprovement may have occurred. Therefore, the overall composite whichthe patient was required to consider, included overall improvement inboth strong and weak odors.

Most patients are usually unconcerned whether or not they can detect orrecognize weak concentrations of odors (DT or RT), but they areconcerned about the intensity (ME) at which odors are perceived. Aftertreatment with 200 mg theophylline, the average improvement in ME forall odors was 5.1% consistent with the results reported with respect tosubjective responses to treatment (Table IV). After treatment with 400mg, mean ME increased 10.4% consistent with increased response to thisdose (Table V) and after treatment with 600 mg, mean ME increased 6.9%(Table VI). These results are more consistent with the subjectiveresponses of the patients, although data for DT and RT increasedsignificantly.

Side effects of theophylline among patients were generally minimal.Patients were required to take the drug in divided doses in middle ofmeals (breakfast and lunch). While this technique delayed drugabsorption it did not inhibit absorption. Thus, the more common andusual side effects of nervousness, jitteriness and difficulty in fallingasleep were obviated. Mild gastrointestinal upset, tachycardia, nausea,diarrhea, headache and insomnia were occasionally reported but wereusually obviated by temporarily decreasing drug intake for a shortperiod and then increasing drug dose to the required amount.

Treatment with theophylline improved smell function probably by actingthrough its effect as a PDE inhibitor on cAMP and cGMP levels in saliva(31) and nasal mucus (34, 35). As drug dose increased, presumably withincreasing PDE inhibition, patient responses also increased. Subsequentincreases in cyclic nucleotides may have increased activation ofolfactory receptor stem cell growth and maturation, as previouslydescribed (20, 21).

This extensive study was performed over an extended time period in aneffort to evaluate treatment of smell loss on the basis of a biochemicalmolecular abnormality as the basic pathology of the loss. Althoughunblinded, a majority of patients expressed subjective improvement intheir sensory function on treatment that was confirmed through objectivetesting.

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Possible novel    mechanism for bitter taste mediated through cGMP. J. Neurophysiol.    1999; 81:1661-5.-   27. Pace, U., Hanski, E., Salomon, Y, et al. Odorant-sensitive    adenylate cyclase may mediate olfactory reception. Nature. 1985;    316:255-8.-   28. Anholt, R. R. H. Molecular neurobiology of olfaction. Crit. Rev.    Neurobiol. 1993; 7:1-22.-   29. Firestein, S., Zufall, F., Sheperd, G. M. Single odor-sensitive    channels in olfactory receptor neurons are also gated by cyclic    nucleotides. J. Neurosci. 1991; 11:3565-72.-   30. Moon, C., Simpson, P. J., Cho, H., et al. Regulation of    intracellular cyclic GMP levels in olfactory sensory neurons. J.    Neurochem. 2005; 95:200-9.-   31. Henkin R. I., Velicu I., Papathanasiu A. cAMP and cGMP in human    parotid saliva: relationships to taste and smell dysfunction, gender    and age. Amer. J. Med. Sci. 2007; 334:431-440.-   32. Henkin, R. I., Velicu, I. cAMP and cGMP in nasal mucus:    relationships to taste and smell dysfunction, gender and age.    Clinical Invest. Med. 2008; 31:E71-E77.-   33. Henkin, R. I. The definition of primary and accessory areas of    olfaction as the basis for a classification of decreased olfactory    acuity, in Olfaction and Taste II, (Hayashi, T. Ed.), Pergamon    Press, London, 1967, pp. 235-252.-   34. Henkin, R. I., Velicu, I. Decreased parotid salivary cyclic    nucleotides related to smell loss severity in patients with taste    and smell dysfunction. Metabolism. 2009; in press.-   35. Henkin, R. I., Velicu, I. cAMP and cGMP in nasal mucus related    to severity of smell loss in patients with smell dysfunction.    Clinical Invest. Med. 2008; 31:E78-E84.-   36. Cai, D., Qiu, J., Cao, Z., McAtee, M., Bregman, B. S.,    Filben, M. T. Neuronal cyclic AMP controls the developmental loss in    ability of axons to regenerate. J. Neurosci. 2001; 21:4731-4739.-   37. Neumann, S., Bradke, F., Tessier-Lavigne, M., Basbaum, A. I.    Regeneration of sensory axons within the injured spinal cord induced    by intraganglionic cAMP elevation. Neuron. 2002; 34:885-893.-   38. Cai, D., Shen, Y., DeBellard, M., Tang, S., Filben, M. T. Prior    exposure to neurotrophins blocks inhibition of axonal regeneration    by MAG and myelin via a cAMP dependent mechanism. Neuron. 1999;    22:89-101.-   39. Kurihara, K., Koyama, N. High activity of adenylyl cyclase in    olfactory and gustatory organs. Biochem. Biophys. Rev. Comm. 1972;    48:30-34.-   40. Pace, U., Hanski, E., Salomon, Y., Lancet, D. Odorant-sensitive    adenylate cyclase may mediate olfactory reception. Nature. 1985;    316:255-258.-   41. Moon, C., Simpson, P. J., Cho, H., Ronnett, G. Y. Regulation of    intracellular cyclic GMP levels in olfactory sensory neurons. J.    Neurochem. 2005; 95:205-209.-   42. Shepherd, G. M. Sensory transduction entering the mainstream of    membrane signaling. Cell. 1991; 67:845-851.-   43. Thompson, W. J. Cyclic nucleotide phosphodiesterase:    pharmacology, biochemistry and function. Pharmacol. Ther. 1991;    51:13-33.-   44. Firestein, B. I., Bredt, D. S. Regulation of sensory neuron    precursor proliferation by cyclic GMP-dependent protein kinase. J.    Neurochem. 1998; 71:1846-1853.-   45. Anholt, R. R. H. Molecular neurobiology of olfaction. Crit. Rev.    Neurobiol. 1993; 7:1-22.-   46. Henkin, R. I., Velicu, I., Papathanasiu, A. Dichotomous changes    in cAMP and cGMP in human parotid saliva after oral theophylline.    FASEB J. 2003; 17:A1028.-   47. Velicu, I., Henkin, R. I. On the antiapoptotic mechanism of    action of theophylline in restoring smell function in patients with    hyposmia. J. Invest. Med. 2005; 53(Suppl. 2):S402.-   48. Levy, L. M., Henkin, R. I., Hutter, A, Lin, C. S.,    Schellinger, D. Increased brain activation in response to odors in    patients with hyposmia after theophylline treatment demonstrated by    fMRI. J. Comp. Asst. Tomog. 1998; 22:760-770.-   49. Henkin, R. I., Velicu, I., Schmidt, L. Effective treatment of    smell loss with theophylline. Exper. Biol. 2008; 22:B976.2.-   50. Henkin, R. I., Larson, A. L., Powell R. D. Hypogeusia,    dysgeusia, hyposmia and dysosmia following influenza-like infection.    Ann. Otol. Rhin. Laryngol. 1975; 84:672-682.-   51. Church, J. A., Bauer, H., Bellanti, J. A., Satterly, R. A.,    Henkin, R. I. Hyposmia associated with atopy. Ann. Aller. 1978;    40:105-109.-   52. Schechter, P. J., Henkin, R. I. Abnormalities of taste and smell    following head trauma. J.

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-   53. Henkin, R. I., Schecter, P. J., Friedewald, W. T., DeMets, D.    L., Raff, M. S. A double blind study of the effects of zinc sulfate    on taste and smell dysfunction. Amer. J. Med. Sci. 1976; 272:    285-299.-   54. Henkin, R. I., Schechter, P. J., Hoye, R. C., Mattern, C. F. T.    Idiopathic hypogeusia with dysgeusia, hyposmia and dysosmia: a new    syndrome. J. Amer. Med. Assoc. 1971; 217:434-440.-   55. Schechter, P. J., Friedwald, W. T., Bronzert, D. A., Raff, M.    S., Henkin, R. I. Idiopathic hypogeusia: a description of the    syndrome and a single blind study with zinc sulfate, in Internat.    Rev. Neurobiol. Suppl. 1., (Pfeiffer, C., Ed.), Academic Press, N Y,    1972, pp. 125-133.-   56. Henkin, R. I. The definition of primary and accessory areas of    olfaction as the basis for a classification of decreased olfactory    acuity, in Olfaction and Taste II, (Hayashi, T. Ed.), Pergamon    Press, London, 1967, pp. 235-252.

Example 2

Theophylline treatment restored smell function in over 50% of thehyposmic patients in Example 1. This study, however, was an open labelclinical trial and not all patients responded to the drug. These resultsraise questions about the character of the study and the efficacy of thedrug to correct the smell loss.

In an effort to understand more about these results, levels of cAMP andcGMP in saliva before and after theophylline treatment were studied inpatients who participated in the clinical study of Example 1. Cyclicnucleotide levels were not assayed until the entire analysis of theclinical trial results was completed.

Methods

Thirty-one patients, aged 29-85 y (56±3 y, Mean±SEM) from among the 312patients who participated in the open label, fixed design clinical trialtreated with theophylline of Example 1 were studied. There were 13 men,aged 54±3 y and 18 women, aged 58±4 y. All patients exhibited hyposmia.Six had Type I hyposmia, 25 had Type II hyposmia. Patients had a varietyof etiologies for their hyposmia; six had PVIL and hypogeusia, thirteenhad allergic rhinitis, nine had a head injury, and three had a varietyof the etiologies contributing to their loss including a drug reaction,an idiopathic cause and post chemotherapy.

Measurements of smell function were obtained for each patient by use ofa standard three stimuli forced choice technique using four odors(pyridine, nitrobenzene, thiophene and amyl acetate as described inExample 1. Subjective measurements of smell function were also obtainedfor each patient by use of standard technique in which smell acuity wasgraded on a scale from 0-100 with 0 indicating an absence of overallsmell function and 100 indicating normal smell function as described inExample 1.

Parotid saliva was collected from each patient by application of aLashley cup over Stensen's duct with lingual stimulation by placement ofconcentrated lemon juice. Saliva was collected in plastic tubes andstored at −20° C. until assayed. cAMP and cGMP were measured in salivaby a sensitive 96 plate spectrophotometric assay (R&D Systems,Minneapolis, Minn.).

All patients were placed in a fixed design open label clinical trialwith treatment with oral theophylline. Treatment consisted of fixedtreatment periods of two-eight months with sequential doses of the drugat 200 mg, 400 mg and 600 mg. At termination of each of these intervals,patients returned to study site for reevaluation. At the end of eachinterval subjective responses to treatment were measured with amodification of the 0-100 scale previously used (vs). Subjectiveresponses to treatment were graded on a sliding scale from −100-0-+100with patients recording improvement (+0-+100), no improvement (0) orworsening (0—-100) of their overall smell function. If overall smellfunction improved ≥5% they were considered to improve clinically (1). Ifsmell function improved <5% they were considered not to improve (1). Atthe end of each interval subjective measurements of smell function,measurements of DT, RT, ME and H and measurements of parotid saliva forlevels of cAMP and cGMP were obtained. In addition, blood plasma wasobtained by venipuncture, placed in ice into zinc free tubes whichcontained 100 mg zinc free heparin, centrifuged at 3000 rpm for 10-20min, the plasma transferred to plastic PCR tubes and stored at −20° C.until assayed. Theophylline was assayed by a fluorescence polarizationassay (Abbott, Chicago, Ill.), as previously described.

At each return visit, if patients noted improvement in their overallsmell function (see Example 1) they continued on this same drug dose andwere not included in any further data. If smell function did not improveon 200 mg of theophylline, their dose was increased to 400 mg and theyreturned to study site after an additional two-four months of treatment.These same measurement processes occurred after treatment with 400 mgand 600 mg of theophylline.

Data for the cyclic nucleotides from these 31 patients was not analyzeduntil the entire study of the 312 patients in Example 1 was assembledand analyzed in its entirety. After completion of these analyses, alldata for parotid saliva cAMP and cGMP from all patients in whom salivarycAMP and cGMP were obtained were assembled. Levels of salivary cyclicnucleotides varied widely. Initially, without external criteria, therewas no way to understand these disparate data. To assist inunderstanding these data, each data point was independently identifiedwith respect to which patient from whom it was obtained and categorizedas to whether or not that patient demonstrated improvement or lack ofimprovement in both subjective smell function and in objectivemeasurements of smell function (DT, RT, ME, H) on theophyllinetreatment. On this basis, 20 patients were identified as having improvedsmell function and 11 did not improve. In a similar manner, measurementsof each patient's plasma theophylline level at each dosage oftheophylline (at 200 mg, 400 mg, 600 mg of theophylline) werecatagorized. The two patient groups were also analyzed post hoc withrespect to type of smell loss and etiology of smell loss.

Differences in the characteristics between these two patient groups wereanalyzed with respect to each measurement obtained (Mean±SEM).Differences between mean±SEM were analyzed using Student t test and X2;differences of p≤5% were considered significant.

Results

Post hoc analysis demonstrated that initial values of subjective smellfunction and measurements of smell function (DT, RT, ME, H) from thesetwo patient groups prior to theophylline treatment did not differ. Atbaseline, there were also no significant differences in saliva cAMP andcGMP between improved and unimproved patients (Table XI). Levels ofsaliva cAMP prior to treatment were consistent with mean values obtainedfor saliva cAMP in all 312 patients included in the original study.After treatment with 200 mg of theophylline, cAMP levels in the improvedpatients increased 10% above baseline, albeit not significantly; therewas essentially no change among the unimproved patients (Table I). Aftertreatment with 400 mg of theophylline, however, although values were notstatistically significant compared to before treatment, cAMP levelsincreased 40% over baseline in the improved group, whereas there wasessentially no change among the unimproved patients. After treatmentwith 600 mg, cAMP levels increased significantly to 67% above initialcAMP values in the improved patients, but there was essentially nochange among unimproved patients. After treatment with 600 mg, levels ofcAMP in the improved patients were still below the mean of saliva cAMPreported in normal subjects.

TABLE XI RESULTS FOR PATIENTS WITH IMPROVED SMELL FUNCTION BEFORETHEOPHYLLINE TREATMENT BEFORE THEOPHYLLINE TREATMENT TREATMENT cAMP(pmol/ml) TREATMENT cGMP (pmol/ml) (pmol/ml) 200 mg 400 mg 600 mg(pmol/ml) 200 mg 400 mg 600 mg 0.87 ± 0.14* 0.96 ± 0.13 1.22 ± 0.331.45^(b) ± 0.37 0.08 ± 0.016 0.08 ± 0.016 0.22^(a) ± 0.07 0.27^(a) ±0.09 (20) (16) (12) (9) (20) (15) (12) (10) PLASMA THEOPHYLLINE (mg/dl)3.5 ± 0.4 6.4 ± 1.2 12.4^(a) ± 1.8 (13) (12) (9) *Mean ± SEM ( ) Patientnumber With respect to no improvement in smell function ^(a)p < 0.05^(b)p < 0.025

At baseline, there were no significant differences between cGMP levelsfor the two groups of patients (Table XII). Mean values obtained in eachpatient group were consistent with values obtained in the total group of312 patients prior to initiation of the treatment (see Example 1). Aftertreatment with 200 mg of theophylline, there was little difference insaliva cGMP levels between improved and unimproved patients or comparedto pretreatment values. After treatment with 400 mg of theophylline,saliva cGMP levels in the improved patients increased significantly to275% over initial values, whereas there was little change among theunimproved patients. After treatment with 600 mg of theophylline, cGMPlevels improved significantly to 338% over initial values, whereas therewas no increase among the unimproved patients. After treatment with 400mg or 600 mg of theophylline, cGMP mean levels in the improved patientswere similar to those obtained in normal subjects.

TABLE XII RESULTS FOR PATIENTS WITH UNIMPROVED SMELL FUNCTION BEFORETHEOPHYLLINE TREATMENT BEFORE THEOPHYLLINE TREATMENT TREATMENT cAMP(pmol/ml) TREATMENT cGMP (pmol/ml) (pmol/ml) 200 mg 400 mg 600 mg(pmol/ml) 200 mg 400 mg 600 mg 1.03 ± 0.24* 0.78 ± 0.26 0.95 ± 0.26 0.56± 0.21 0.08 ± 0.016 0.07 ± 0.018 0.05 ± 0.014 0.04 ± 0.07 (11) (9) (5)(9) (11) (9) (4) (5) PLASMA THEOPHYLLINE (mg/dl) 3.7 ± 1.0 8.6 ± 1.4 8.7± 0.6 (9) (5) (9) *Mean ± SEM ( ) Patient number

Comparison of blood plasma theophylline between the two groups indicatedthat there was no difference in mean values after treatment with either200 mg or 400 mg of theophylline (Table XI). After treatment with 600mg, however, although plasma theophylline increased in both groups,compared to plasma levels seen following treatment with 200 mg or 400 mgof theophylline, mean theophylline was significantly elevated in theimproved group compared to the unimproved group. (Tables XI and XII).

Classified by smell loss type, there were significant differencesbetween the two groups (Table XIII). Among the improved patients, 95% ofthe patients exhibited Type II hyposmia, whereas, only one patient hadType I hyposmia. Among the unimproved patients, 57% of the patientsexhibited Type I hyposmia, indicating that significantly more of theunimproved patients (with unchanged levels of cAMP and cGMP) had Type Ihyposmia, the more severe type of smell loss (Table XII).

TABLE XIII COMPARISON OF CLINICAL CHARACTERISTICS BETWEEN IMPROVED ANDUNIMPROVED PATIENTS IMPROVED* UNIMPROVED SMELL LOSS TYPE I II I IIPATIENT NUMBER 1 19^(a) 4 7 ETIOLOGY IMPROVED* UNIMPROVED OF LOSS ARPIHH HI OTHER AR PIHH HI OTHER PATIENT 8 6 3 3 8 0 3 0 NUMBER *Parotidsaliva cAMP and cGMP concentrations after 600 mg theophylline weresignificantly higher in improved than in unimproved patients (see TablesI, II) Improved vs Unimproved by Smell Loss Type ^(a)X² = 5.2 (p < 0.05)AR, allergic rhinitis PIHH, post influenza-like hyposmia and hypogeusiaHI, head injury Other (drug reaction, idiopathic, post chemotherapy)

Classified by diagnosis, the distribution of etiology of smell lossamong the improved patients was similar to that previously reported inseveral publications. Among the unimproved patients, however, there wereno patients who had PIHH or “other” causes of smell loss. Indeed, onlytwo etiologies comprised the etiology of smell loss among unimprovedpatients, either allergic rhinitis or head injury (Table XII).

DISCUSSION

The observed results were unexpected. These results, however, suggestthat changes in cGMP may be more relevant to changes in smell functionthan measurement of cAMP since, among the improved patients, levels ofcGMP increased into the normal range whereas for cAMP they increased,but not into the normal range.

These results also suggest that administration of theophylline at thesame dose results in differences in smell improvement, in levels ofsaliva cAMP or cGMP and in serum theophylline levels in this group ofpatients. There are no prior studies that would suggest differentialeffects of drug response or biochemical changes related to theophyllineintake. This type of change, however, has been observed previously withmany drugs and is well known in several disease states. Indeed, drugresistance has been important to understanding differential drug affectsand their influence on metabolic processes. Although the patient numberin this study is relatively small, apparently this is the first reportof drug resistance to oral administration of theophylline.

Patients with theophylline resistance appear to have specific clinicalcharacteristics by which they can be identified prior to their treatmentand development of this resistance. It was discovered that unimprovedpatients had head injury and allergic rhinitis. In addition thesepatients exhibit a preponderance of a severe type of smell loss (Type Ihyposmia), one in which they cannot recognize the character of any odor.

The data obtained also suggest that this resistance is presentirrespective of drug dose, although it is more obvious at higher dosesof the drug. In addition, drug resistance effects appear to be morerobust with respect to levels of saliva cGMP rather than to cAMP. Levelsof salivary cGMP among the improved patients returned to levelspreviously observed in normal subjects. Saliva cAMP levels increased,significantly so at the highest dose of theophylline administered, butthey did not increase to levels measured in normal subjects.

While levels of blood theophylline increased in both groups of patientsas the dose of theophylline increased (200 mg, 400 mg, 600 mg), levelsof blood theophylline were significantly higher in the improved groupcompared to the unimproved group after 600 mg of the drug. The bloodtheophylline level in the improved group was 12.4 mg/dl whereas it was8.7 mg in the unimproved group (Tables XI and XII). While the level oftheophylline in the improved patients is in the normal range fortherapeutic effects of the drug (10-20 mg/dl), the level in theunimproved group was not. Previous data suggest that patients thatimprove with theophylline exhibit plasma levels between 2-14 mg/dl, sothat it would not be reasonable to imply that these differences betweenthese two groups are attributable only to the differences obtained inserum theophylline. While this is undoubtedly one factor related to drugresistance, as is well known from many other studies, this differencemay not account for all the differences between these two groups.

The open label trial of theophylline in Example 1 demonstrates theusefulness of theophylline in restoring the sense of smell in patientswith smell loss. The present studies indicate that multiple factorsinfluence successful treatment of hyposmic patients and that a thoroughwork up is required if treatment is to be successful. These factorsinclude testing as to the type and degree of smell loss, discovering theetiology of the patient's smell loss, analysis of theophylline plasmalevels, and analysis of parotid gland secretion of cAMP and cGMP. Inparticular, parotid saliva cGMP level stands out as predictive ofclinical response given the unexpected finding that cGMP levelscorrelate with recovery of the sense of smell. This observation allowsfor the development of a testing regiment to select for the appropriatetherapy. A patient can be administered a standard challenge dose oftheophylline or other PDE inhibitor and then the level of parotid salivacGMP is determined. Patients that achieve a threshold level of cGMPfollowing the challenge can then be prescribed the appropriate dose thatwill achieve the targeted steady state plasma level of the PDEinhibitor. Patients whom parotid saliva cGMP levels fail to achieve thethreshold level can be challenged with a higher dose of the PDEinhibitor, have another PDE inhibitor or other active agent added to thedosage of the original PDE inhibitor, or switched to another PDEinhibitor as is clinically warranted. In this way, a physician candetermine the optimum PDE inhibitor dosage for a patient withoutundertaking the long dose escalation titration required in the originalstudy detailed in Example 1.

In some embodiments, a method is provided for screening patients for PDEinhibitor therapy for anosmia or hyposmia comprising: administering to apatient a challenge dose of a PDE inhibitor; determining the salivarylevel of cGMP; and comparing the patient's salivary cGMP level to athreshold value, wherein patients who have a cGMP salivary level equalto or greater than the threshold value are candidates for PDE inhibitortherapy to treat anosmia or hyposmia. In some embodiments, the challengedose is 200 mg, 400 mg, 600 mg or 800 mg of theophylline. In someembodiments, the threshold value is at least 0.08, 0.10, 0.12, 0.14,0.16, 0.18, 0.20, 0.22, 0.24, 0.26, or 0.28 pmol/ml.

In some embodiments, the threshold value is equal to or substantiallysimilar to the mean cGMP value seen in normal individuals following thesame challenge dose. In other embodiments, the threshold value is notless than 10%, 20%, 30%, 40%, or 50% of the mean salivary cGMP valueseen in normal individuals following the same challenge dose.

Example 3

Hyposmic patients for whom the etiology of smell loss is not associatedwith head injury or allergic rhinitis are enrolled in an open labeltrial of inhalable theophylline. Patients are started at an initial doseof not more than 500 μg formulated as a liquid spray or dry powder to bedelivered as a metered dose. Initially only local patients are enrolledso that quicker dose titration is achieved. Groups of 5 patients arestarted on not more than 500 μg of theophylline and continued for 1month.

Patients are tested for improvement in smell acuity using the standardforced choice technique using four odors as described in Example 1.Additionally, subjective measurements of smell function are obtainedusing a scale from 0-100 as described in Example 1. Blood samples arealso drawn to ascertain blood theophylline level to gauge itsrelationship to the expected recovery of smell.

Nasal administration of theophylline provides for high, initialconcentrations of theophylline being deposited on the olfactoryepithelium, thereby exposing olfactory neurons and their sensory ciliato higher concentrations of theophylline than are achieved through oraladministration through the avoidance of first pass metabolism. Nasaladministration provides for at least an equivalent recovery of smellacuity comparable to that achieved with orally administered theophyllinewhile avoiding or at least reducing the side effects caused by highertheophylline plasma levels seen with oral administration.

Example 4

Hyposmic patients for whom the etiology of smell loss is not associatedwith head injury or allergic rhinitis are enrolled in an open labeltrial of an inhalable PDE1 inhibitor. More preferably, a PDE1C2inhibitor such as eburnamenine-14-carboxylic acid ethyl ester(vinpocetine), 8-methoxymethyl-1-methyl-3-(2-methylpropyl) xanthine(8MM-IBMX), zaprinast (M&B 22948),4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram),4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724),1,6-dihydro-2-methyl-6-oxo-(3,4′-bipyridine)-5-carbonitrile (milrinone),trequinsin (HL 725), and/or combinations thereof because of the highexpression level of PDE1C2 in the olfactory epithelium. Patients arestarted at an initial dose of 500 μg formulated as a liquid spray or drypowder delivered as a metered dose. Initially only local patients areenrolled so that quicker dose titration is achieved. Groups of 5patients are started on 500 μg of the PDE1 inhibitor or PDE1C2 inhibitorand continued for 1 month.

Patients are tested for improvement in smell acuity using the standardforced choice technique using four odors as described in Example 1.Additionally, subjective measurements of smell function are obtainedusing a scale from 0-100 as described in Example 1. Blood samples arealso drawn to ascertain blood levels of the PDE1 inhibitor or PDE1C2inhibitor and its relationship to the observed patient response.

Nasal administration of the PDE1 inhibitor or PDE1C2 inhibitor provideshigher exposure of the PDE1 inhibitor or PDE1C2 inhibitor to nasalolfactory neurons than can be achieved through oral administration. Thisprovides for at least an equivalent recovery of smell acuity comparableto that achieved with orally administered PDE1 inhibitor or PDE1C2inhibitor, avoiding or reducing the side effects caused by higher PDE1inhibitor or PDE1C2 inhibitor plasma levels seen with oraladministration.

Example 5

For over 60 years, theophylline has been used as a bronchodilator in thetreatment of asthma and COPD and remains one of the most widelyprescribed drugs for the treatment of airway diseases worldwide.Theophylline directly relaxes human airways smooth muscle in vitro and,like beta₂-agonists, acts as a functional antagonist, preventing andreversing the effects of all bronchoconstrictor agonists.Bronchodilatation by theophylline is achieved through PDE inhibition,resulting in an increase in cAMP by inhibition of PDE3 and PDE4 and incyclic guanosine 3′,5′-monophosphate by inhibition of PDE5. Thebronchodilator effect of theophylline in human airways is reduced bycharybdotoxin, which selectively inhibits large conductance Ca²⁺activated K⁺ channels (maxi-K channels), suggesting that theophyllineopens these maxi-K channels via an increase in cAMP.

In asthma therapy, theophylline has an increasing acute bronchodilatorresponse above plasma concentrations of 10 mg/L (55 μM), however, theupper recommended plasma concentration is 20 mg/L due to unacceptableside effects above this level including nausea and headaches. Thetherapeutic range for plasma concentrations is therefore established at10 to 20 mg/L, and doses are adjusted in individual patients to achievethis range.

Theophylline has an additional effect on mucociliary clearance through astimulatory effect on ciliary beat frequency and water transport acrossthe airway epithelium. Relatively high doses of theophylline are needed,as this effect is likely to be due to an increase in cAMP as a result ofPDE inhibition.

Theophylline also has anti-inflammatory effects in asthma. In allergenchallenge studies in patients with asthma, intravenous theophyllineinhibits the late response to allergen. A similar finding with allergenchallenge was reported after chronic oral treatment with theophylline.Oral theophylline also inhibits the late response to toluenediisocyanate in toluene diisocyanate-sensitive individuals with asthma.This is interpreted as an effect on the chronic inflammatory response,and this is supported by a reduced infiltration of eosinophils and CD4⁺lymphocytes into the airways after allergen challenge subsequent to lowdoses of theophylline. In patients with nocturnal asthma, low-dosetheophylline inhibits the influx of neutrophils and, to a lesser extent,eosinophils in the early morning. In patients with mild asthma, lowdoses of theophylline (mean plasma concentration ˜5 mg/L) reduce thenumbers of eosinophils in bronchial biopsies, bronchoalveolar lavage,and induced sputum, whereas in severe asthma, withdrawal of theophyllineresults in increased numbers of activated CD4⁺ cells and eosinophils inbronchial biopsies.

In patients with COPD, theophylline reduces the total number andproportion of neutrophils in induced sputum, the concentration ofinterleukin-8, and neutrophil chemotactic responses, suggesting anantiinflammatory effect. This is in sharp contrast to the lack of effectof high doses of inhaled corticosteroids in a similar population ofpatients.

These anti-inflammatory effects of theophylline in asthma and COPD areseen at concentrations that are usually less than 10 mg/L, which isbelow the dose where significant clinically useful bronchodilatation isevident.

Theophylline is a weak and nonselective inhibitor of PDEs. In vitro,theophylline relaxes airway smooth muscle by inhibition of PDE activity(PDE3, PDE4, and PDE5), but relatively high concentrations are neededfor maximal relaxation. The degree of PDE inhibition is very small atconcentrations of theophylline that are therapeutically relevant withexperiments with human lung extracts demonstrating only 5 to 10%inhibition of total PDE activity at therapeutic concentrations.

Inhalation therapy with neublized solutions of theophylline and othermethylxanthines was tried to widen the therapeutic index. Inhalation ofmethylxanthine derivatives produce an immediate increase in specificairway resistance (sGaw) that peaks in 5 minutes, but the effects wereno more than half the response seen with a standard 200 μs dose ofinhaled salbutamol, and dissipated by 30 minutes. Additionally, thenebulized solutions of methylxanthine derivatives have a disagreeabletaste and produced a pronounced cough leading the researchers toconclude that inhalation of methylxanthines derivatives were unlikely tobe of benefit in the treatment of asthma.

Applicants believe that this past failure with inhalable methylxanthinederivatives owes more to the formulation and method of delivery than tothe inherent properties of the derivatives. To test this theory,asthmatic patients are enrolled in a dose escalating single blind study.On arrival to the clinic, subjects are allowed to rest for 20 minutesbefore baseline measurements of FEV1 (six recordings) and sGaw (fiverecordings) are made. Subjects receive a dry powder dispenser loadedwith a dose of 2 mg theophylline or a dry powder carrier. Subjects areadvised that some of the preparations may have a bitter taste, whileothers will not, but that this does not necessarily reflect the presenceor degree of activity of the drug. After each inhalation, measurementsof sGaw are made at 1, 3, 5, 10, 15, 20, 25, and 30 minutes. After 30minutes, the sGaw measurement is still 20% above the baselinemeasurement, so additional sGaw measurements are taken every 15 minutesuntil baseline is reached at 1 hour.

On completion of the last recording, subjects inhale a 200 μg dose ofsalbutamol from a metered inhaler and a further measurement of sGaw ismade after 15 minutes. Subjects are also monitored for coughing and arequestioned about the taste of the drug. The increase in sGaw produced bythe inhaled theophylline is comparable to that produced by salbutamol.Furthermore, the dry powder formulation is without the unpleasant tasteor the induction of coughing associated with a liquid formulationdelivered by a nebulizer.

Example 6

IBMX, (isobutylmethylxanthine), a non-specific PDE inhibitor possessesgreater potency than theophylline (IC50=2-50 μM). Given IBMX structuralsimilarity to theophylline, IBMX shares theophylline's bronchodialation,anti-inflammatory, and ciliary beat frequency stimulatory effects but isexpected to have a wider therapeutic index potentially allowing for theuse of lower dosages, thereby reducing side effects and complaints overdisagreeable taste when administered through a nebulizer.

Asthmatic patients are enrolled in a dose escalating single blind study.On arrival to the clinic, subjects are allowed to rest for 20 minutesbefore baseline measurements of FEV1 (six recordings) and sGaw (fiverecordings) are made. Subjects receive a dry powder dispenser loadedwith a dose of 1 mg IBMX formulated as a dry powder or a dry powdercarrier. Subjects are advised that some of the preparations may have abitter taste, while others will not, but that this does not necessarilyreflect the presence or degree of activity of the drug. After eachinhalation, measurements of sGaw are made at 1, 3, 5, 10, 15, 20, 25,and 30 minutes. After 30 minutes, the sGaw measurement is still 20%above the baseline measurement, so additional sGaw measurements aretaken every 15 minutes until baseline is reached at one and a halfhours.

On completion of the last recording, subjects inhale a 200 μg dose ofsalbutamol from a metered inhaler and a further measurement of sGaw madafter 15 minutes. Subjects are also be monitored for coughing and arequestioned about the taste of the drug. The increase in sGaw produced bythe inhaled IBMX is comparable with that produced by salbutamol.Furthermore, the dry powder formulation is without the unpleasant tasteor the induction of coughing associated with a liquid formulationdelivered by a nebulizer.

Example 7

Ten patients with hyposmia were selected from among 400 patients withhyposmia to participate in a pilot study to determine safety andefficacy of intranasal theophylline for the treatment of hyposmia. Thesepatients were previously treated with oral theophylline or were switchedfrom oral theophylline to intranasal theophylline at the start of thestudy. Selection for inclusion in the intranasal study was based uponseveral criteria. 1) non-response to oral theophylline; 2) severe sideeffects with oral theophylline that prevented reaching a dose ofsufficient strength to restore smell function; and/or 3) preference forintranasal medication over oral medication.

Through careful evaluation of plasma, saliva and nasal mucustheophylline levels in patients with hyposmia taking theophylline atdoses of 200-800 mg daily, it was determined that a dose of 20 μgtheophylline delivered intranasally to each naris (or 40 μg total) wassufficient to produce localized effects similar to those achieve withoral theophylline of 200-400 mg daily. Additionally, loco-regionaladministration of such a low dose would avoid producing the side effectsseen with oral administration.

Intranasal Preparation

A batch of theophylline for intranasal administration at a dose of 20μg/0.4 ml of was prepared by dissolving 250 mg of methylparaben powderand 250 mg of propylparaben powder in 5 ml of propylene glycol. Next 50mg of theophylline, anhydrous powder, was dissolved in a small amount of0.9% sodium chloride. The dissolved parabens were added to thetheophylline solution and mixed well. Sufficient 0.9% sodium chloridewas added to the mixture to bring the total volume to 1000 ml. Thesolution was sterilized by filtering through a sterile 0.2 μm filter. 1ml syringes were loaded with 0.4 ml of the sterile solution and thencapped with a tamper evident cap. Representative samples were sent to anindependent testing laboratory for pH, endotoxin, sterility, and fungalcontamination testing in addition to the determination of thetheophylline concentration. The test results demonstrated that thepreparation had 20.716 μg of theophylline per 0.4 ml with a pH of 5.9.Further, endotoxin was below 1.0 EU/ml and the preparation was sterileand free of fungal contamination.

Study Design

The purpose, risk and benefits of participating in the study wereexplained to each patient by the study's supervising physician. Prior toenrollment in the study, each patient read and signed an informedconsent form. Next, each patient was instructed in the proper techniqueof intranasal administration.

Patients discontinued their use of oral theophylline either at time ofentry into the study (eight patients) or four months prior to studyentry (two patients).

Changes to smell function was determined at four specific periods:

Time 0—at the start of the study.

Time 1—one week after starting intranasal theophylline

Time 2—two weeks after starting intranasal theophylline

Time 4—four weeks after starting intranasal theophylline

After reaching Time 4, the study was discontinued and the patients werereturned to their use of oral theophylline if indicated.

Study Measurements

At each of the four time periods of the study the following battery oftests were performed and samples obtained.

Objective Test Measurements

Taste Function Tests

Taste function was measured by detection threshold (DT), recognitionthreshold (RT), magnitude estimation (ME) and hedonics (H) for fourtastants (NaCl, sucrose, HCl, urea) by use of a standard three stimuli,forced choice staircase drop technique described in Example 1 and ref53.

Smell Function Tests

Smell function was measured for DT, RT, ME and H for four odorants(pyridine, nitrobenzene, thiophene, amyl acetate) by use of the standardthree stimuli, forced choice staircase sniff technique described inExample 1 and in ref 53.

Bodily Fluids

Blood was obtained by venipuncture to collect plasma and red blood cellsused to measure trace metals (Cu, Zn, Mg), various enzymes, theophyllineand other chemical moieties.

Saliva was collected with a modified Lashley cup as described in Example2 and used to measure trace metals (Cu, Zn, Mg), various enzymes (cAMP,cGMP, CA VI, etc.), theophylline and other chemical moieties.

Nasal mucus was collected by use of a standard technique and used tomeasure trace metals (Cu, Zn, Mg), various enzymes (cAMP, cGMP, CA VI,etc.), theophylline and other chemical moieties. Trace metals weremeasured by atomic absorption spectrophotometry (as previouslydescribed) using a dual beam Thermo-Jarrel Ash atomic absorptionspectrophotometer. CA VI was measured by enzymatic analysis of activityof the enzyme by our modification of the method of Richli, et al. Cyclicnucleotides were measured by a sensitive 96 plate spectrophotometricsensitive ELISA assay provided by Applied Biosystems, Minneapolis, Minn.

Subjective Test Measurements

Subjective responses to treatment were obtained independent of anyinteraction with the clinical staff by using a scale of 0-100 to measureresponse of taste function and smell function to treatment with 0indicating no response, 100 indicating return to normal function andintermediate numbers indicating an intermediate response.

Treatment Technique

The patients inserted a plastic syringe containing 0.4 ml of fluid (20μg theophylline) into each naris once a day. Previous investigation ofthe syringe injection technique for intranasal drug administrationindicated that a volume of 0.4 ml delivered to each naris was sufficientto deliver the drug dose without having the liquid escape out of thenares or directly into the pharynx. The drug dose was delivered througha plastic nipple which fitted snugly onto the filled syringe which wasattached directly to the nipple. One nipple was supplied for each set oftwo syringes in each application kit. The patient fitted the nipple onone syringe, placed the nipple securely into the lower naris andinjected the contents of the syringe directly into the lower vault ofone naris accompanied by a modest inhalation. This technique was thenused for the second syringe used for the other naris. The patients wereinstructed to administer the drug either seated or standing with theirhead in an erect, vertical position.

Patients were supplied with 15 doses on each of two occasions with allused syringes returned to the clinic after use to insure proper andcomplete usage.

Results

Ten hyposmia patients were enrolled in the study, eight patients havecompleted all four phases of the study with the remaining two patientsstill in process. No side effects of intranasal use were observed,including, no nasal congestion, nasal pruritus, nasal discomfort, coughor unusual or bitter taste. No patient exhibited a further of loss ofsmell while in the study. This demonstrates that the patients thatstopped their oral medication at the time of the start of the study,which ranged from 400-800 mg oral theophylline daily, received at leastan equivalent localized dose of theophylline to the nasal olfactoryepithelium as was achieved with their total oral dose.

Of the eight patients that completed the study, six reported subjectiveimprovement in both taste and smell function (>10-20% over prior resultswith oral theophylline). The plasma theophylline levels for the eightpatients was zero indicating that at most an undetectable fraction ofintranasal theophylline was absorbed systemically. Furthermore, thisdata demonstrates that at the dosage level of 20 μg per naris, moretheophylline is delivered locally to the nasal olfactory epithelium thanis delivered by 200-800 mg of oral theophylline because the six patientsthat experienced increased smell acuity with the intranasal theophyllinereported that their acuity diminished upon the resumption of theirprevious oral dosage of theophylline.

Overall, of the first eight patients, six (75%) exhibited subjectiveimprovement in smell function in the study with the other two patientsreporting maintenance of the improvement in smell acuity previouslyachieved with oral theophylline.

Discussion

Preliminary results of this trial of intranasal theophylline indicatethat patients can administer the drug without difficulty. Further, noside effects were observed with the intranasal administration oftheophylline. Additionally, all of the patients preferred intranasaladministration over oral administration with six of the eight evaluablepatients studied noting significant improvement in taste and smellfunction after use of intranasal over that achieved with oraltheophylline. All ten patients reported no diminution of their smellfunction using intranasal administration demonstrating that they arereceiving at least an equivalent dose of theophylline by intranasaladministration. As the last two patients have not yet completed thestudy, the number of patients responding to the intranasal therapy mayfurther increase. Furthermore, taste and smell acuity was stable orimproved without producing measureable blood theophylline levels.

One of the patients that achieved an improved sense of smell did notwish to return the unused portion of the intranasal theophylline sincethe methodology also successfully enhanced her nasal breathing, nasalhomeostasis and ability to sleep more soundly at night due toimprovement in nasal function.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1.-17. (canceled)
 18. A method of treating or preventing anosmia orhyposmia comprising administering to a subject having the anosmia orhyposmia a pharmaceutical dosage unit comprising a phosphodiesterase(PDE) inhibitor, wherein the administering is sufficient to increase alevel of cAMP or cGMP in nasal mucus or saliva in the subject by atleast about 10%, relative to a level of cAMP or cGMP prior to theadministering.
 19. The method of claim 18, wherein the PDE inhibitorcomprises theophylline.
 20. The method of claim 18, wherein the dosageunit comprises a positive amount of: less than 500 μg, less than 250 μg,or less than 100 μg of the PDE inhibitor.
 21. The method of claim 18,wherein the dosage unit is formulated as a liquid.
 22. The method ofclaim 21, wherein the pH of the dosage unit is from about 5.5-7.5. 23.The method of claim 18, wherein the dosage unit is formulated as a drypowder.
 24. The method of claim 18, wherein the dosage unit isadministered at least once, twice, or thrice daily.
 25. The method ofclaim 18, wherein the administering is sufficient to increase taste orsmell acuity by at least 5%.
 26. A method of treating or preventinganosmia or hyposmia comprising administering to a subject having theanosmia or hyposmia a pharmaceutical dosage unit comprising a PDEinhibitor, wherein the administering is sufficient to decrease Hedonic(H) value for pyridine or thiophene and increase H value fornitrobenzene and amyl acetate while increasing Magnitude Estimation (ME)values for pyridine, thiophene, nitrobenzene, or amyl acetate.
 27. Themethod of claim 26, wherein the PDE inhibitor comprises theophylline.28. The method of claim 26, wherein the dosage unit comprises a positiveamount of: less than 500 less than 250 or less than 100 μs of the PDEinhibitor.
 29. The method of claim 26, wherein the dosage unit isformulated as a liquid.
 30. The method of claim 29, wherein the pH ofthe dosage unit is from about 5.5-7.5.
 31. The method of claim 26,wherein the dosage unit is formulated as a dry powder.
 32. The method ofclaim 26, wherein the dosage unit is administered at least once, twice,or thrice daily.
 33. The method of claim 26, wherein successfultreatment comprises an increase in taste or smell acuity of at least 5%.34. A method of treating or preventing anosmia or hyposmia comprisingadministering to a subject having the anosmia or hyposmia apharmaceutical dosage unit comprising theophylline, wherein theadministering is sufficient to provide a concentration of theophyllinein nasal mucus sufficient to; a. treat taste or smell loss, b. increasecAMP or cGMP, or c. both.
 35. The method of claim 34, wherein the dosageunit comprises a positive amount of: less than 500 μg, less than 250 μg,or less than 100 μs of the theophylline.
 36. The method of claim 34,wherein the dosage unit is formulated as a liquid.
 37. The method ofclaim 36, wherein the volume of the dosage unit is about 0.4 mL.
 38. Themethod of claim 36, wherein the pH of the dosage unit is from about5.5-7.5.
 39. The method of claim 34, wherein the dosage unit isformulated as a dry powder.
 40. The method of claim 34, wherein thedosage unit is administered at least once, twice, or thrice daily. 41.The method of claim 34, wherein after a period of daily administrationlasting at least a month, taste or smell acuity improves by at least 5%.42. A method of treating or preventing anosmia or hyposmia in a subjectin need thereof comprising intranasally administering to the subject adosage unit comprising a PDE inhibitor, wherein the PDE inhibitor ispresent in serum at a concentration of no more than 1 mg/dL, asdetermined by a fluorescence polarization assay.